Method for purifying antibodies and compositions thereof

Abstract

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

Description

[Technical Field]

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

[0002] Related applications This application is U.S. Provisional Application No. 62 / 859,580, filed on June 10, 2019. Priority is claimed based on the above application. The entirety of the above application is incorporated herein by reference. ru.

[0003] Sequence List This application includes a sequence listing submitted electronically in ASCII format, and the entire contents thereof are referenced. This is incorporated herein by reference. The aforementioned ASCII copy was created on June 5, 2020. It is named T103022_1120WO_SL.txt and its size It is 10,015 bytes. [Background technology]

[0004] Large-scale, economical protein purification is becoming an increasingly important task in the biotechnology industry. This is becoming a topic of discussion. Generally, biologics produce large quantities of the target therapeutic protein. Manipulated cells, such as prokaryotic cell lines like bacterial cells, or mammalian or fungal cells. It is produced by cell culture using eukaryotic cell lines such as the following. The cell lines used are living organisms. Therefore, it contains sugars, amino acids, and, in some cases, growth factors supplied from animal serum preparations. A complex cell culture medium must be supplied. The desired recombinant therapeutic protein, for example, , cell culture medium components, host cell proteins (HCPs), host nucleic acids, and / or chromatographs Process-related impurities such as feed materials, as well as aggregates, misfolded chemical species, Alternatively, product-related impurities such as fragments of the target protein can be used as therapeutic agents for humans. Achieving sufficient purity through separation was a challenging task.

[0005] Product-related and process-related impurities, including aggregates, interfere with the purification process and maintain the It may affect proteins during administration, and / or antibodies may be targeted as pharmaceuticals. There is a risk of adverse reactions when administered (Shukla et al., J. C hromatogr.B.Analyt.Technol.Biomed.Life S ci., 848(1), 28-39).

[0006] Therefore, to effectively remove impurities, improve protein recovery rates, and meet therapeutic requirements An improved method for purifying therapeutic proteins, such as antibodies, while maintaining optimal conditions. This remains in demand in the relevant technological field. [Overview of the project]

[0007] The present invention relates to a method for purifying anti-α4β7 antibodies, such as vedolizumab, from a liquid solution, for example. We will specifically provide a method.

[0008] In one embodiment, the present invention relates to a liquid solution containing an anti-α4β7 antibody and one or more impurities, from which an anti-α A method for obtaining a composition containing a 4β7 antibody, wherein a matrix containing protein A is used. The anti-α4β7 antibody is brought into contact with a liquid solution containing anti-α4β7 antibody and one or more impurities, thereby purging the anti-α4β7 antibody. The process involves binding to rothein A and washing the matrix containing protein A with a washing solution. Furthermore, by contacting the matrix with an elution solution with a pH of 3.2-4, protein A can be extracted. An anti-α4β7 antibody is eluted from the matrix to obtain a composition containing the anti-α4β7 antibody. comprising, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and has the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2 in the heavy chain variable region, and the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6 in the light chain variable region, characterized by a method.

[0009] In one embodiment, the method is used to obtain a composition containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α 4β7 antibody and one or more impurities. The method comprises contacting a matrix containing protein A with a liquid solution containing an anti-α4β7 antibody and one or more impurities to bind the anti-α4β7 antibody to protein A, washing the matrix with a washing solution containing protein A, and eluting the anti-α4β7 antibody from the matrix containing protein A by contacting the matrix with an elution solution having a pH of 3.2 to 4, thereby obtaining a composition containing less than 1% HMW aggregates. In one embodiment, the protein A is immobilized on a solid phase. In one embodiment, the solid phase comprises one or more of beads, gels, and resins.

[0010] In one embodiment, the washing solution has a pH of about 7. In one embodiment, the elution solution contains citric acid.

[0011] In one embodiment, the elution solution has a pH of 3.2 to 3.7.

[0012]

[0013] In another aspect, the present invention provides an anti-α4β7 antibody and one or more impurities from a liquid solution containing an anti- ​​​​​​​​​​A method for obtaining a composition containing an α4β7 antibody, comprising an anti-α4β7 antibody and at least one A solution containing two impurities is mixed with a hydrophobic interaction chromatography (HIC) resin and HIC By contacting the resin under conditions that allow for the flow-through of the anti-α4β7 antibody, A composition containing an anti-α4β7 antibody was obtained, and the HIC resin was characterized as a highly hydrophobic HIC resin. The anti-α4β7 antibody is a humanized antibody, specifically an IgG1 antibody, as described in Sequence ID No. 4. The CDR3 domain, the CDR2 domain described in Sequence ID No. 3, and the C described in Sequence ID No. 2 It includes a heavy chain variable region containing the DR1 domain, and the CDR3 domain described in Sequence ID No. 8. , including the CDR2 domain described in Sequence ID No. 7 and the CDR1 domain described in Sequence ID No. 6 The method is characterized by including a variable light chain region.

[0014] In one embodiment, the method involves using a liquid solution containing an anti-α4β7 antibody and one or more impurities. This is for obtaining a composition containing an anti-α4β7 antibody and less than 0.6% HMW aggregates. The above method involves hydrophobic interaction between a solution containing the anti-α4β7 antibody and at least one impurity. Action chromatography (HIC) resin and flow of anti-α4β7 antibody through the HIC resin. By bringing the samples into contact under conditions that allow pass-through, the anti-α4β7 antibody and 0.6% untreated A composition containing a full HMW aggregate is obtained, and the anti-α4β7 antibody is a humanized antibody, IgG It is an antibody containing the CDR3 domain described in SEQ ID NO: 4 and the CDR2 domain described in SEQ ID NO: 3. The sequence includes a heavy chain variable region containing the CDR1 domain described in Sequence ID No. 2, and The CDR3 domain described in number 8, the CDR2 domain described in sequence number 7, and sequence number It includes a light chain variable region containing the CDR1 domain described in 6.

[0015] In one embodiment, the HIC resin is leveled in a phosphate buffer having a pH of less than approximately 7.2. It is tempered. In one embodiment, the phosphate buffer is about 0.35 mM to about 0.15 mM It contains potassium phosphate.

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

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

[0018] In one embodiment, the composition contains less than approximately 0.3 ppm of host cell proteins (HCPs). To possess.

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

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

[0021] In another aspect, the present invention relates to a liquid solution containing an anti-α4β7 antibody and one or more impurities from which an anti- A method for producing a preparation containing an α4β7 antibody, comprising an anti-α4β7 antibody and one or more By contacting a liquid solution containing impurities with a mixed-mode chromatography resin, the anti- The process involves binding the α4β7 antibody to the resin and washing the mixed mode chromatography resin with a washing solution. Washing with this method and bringing the resin into contact with an elution solution having a pH of 3.9 or higher The anti-α4β7 antibody was purified by eluting it from the mixed-mode chromatography resin. The objective is to obtain a formulation containing an anti-α4β7 antibody, and that the anti-α4β7 antibody is the same as described in SEQ ID NO: 1. The method is characterized by including a chain-variable region and a light chain-variable region as described in Sequence ID No. 2.

[0022] In one embodiment of the above-described model, the method comprises a liquid containing an anti-α4β7 antibody and one or more impurities. This is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a body solution, and The method involves mixing-mode chromatography of a liquid solution containing an anti-α4β7 antibody and one or more impurities. By contacting the resin with the Raffy resin, the anti-α4β7 antibody is bound to the resin, and The mixed mode chromatography resin is washed with the washing solution, and the pH is 3.9 or higher. Mixed-mode chromatography is performed by contacting the resin with an elution solution having the above pH. By eluting the anti-α4β7 antibody from the resin, a formulation containing less than 1% HMW aggregates is obtained. This includes obtaining [something].

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

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

[0025] In some embodiments, the elution solution is NaCl with a concentration of approximately 160 mM to approximately 240 mM. Includes.

[0026] In certain embodiments, the mixed-mode chromatography resin is CaptoAdher It is e ImpRes.

[0027] In some embodiments of the above-described model, the method uses a cation exchange (CEX) resin. The method further includes purifying the anti-α4β7 antibody. In some such embodiments, the CEX tree Lipids are manipulated in binding / elution mode.

[0028] In another aspect, the present invention relates to a liquid solution containing an anti-α4β7 antibody and one or more impurities from which an anti- A method for producing a preparation containing an α4β7 antibody, comprising an anti-α4β7 antibody and one or more By contacting a liquid solution containing impurities with a mixed-mode chromatography resin, the anti- The process involves binding the α4β7 antibody to the resin and washing the mixed mode chromatography resin with a washing solution. Washing with a solution having a pH of 4.2 or lower and an elution rate of 28 mS / cm or lower. By bringing the solution into contact with the resin, anti-α4β7 antibodies are obtained from the mixed-mode chromatography resin. By dissolving the body, a formulation containing purified anti-α4β7 antibody is obtained, and anti-α4 The β7 antibody interacts with the heavy chain variable region described in SEQ ID NO: 1 and the light chain variable region described in SEQ ID NO: 2. It includes, and is characterized by, a method.

[0029] In some embodiments of the above-described model, the method includes an anti-α4β7 antibody and one or more impurities. This is for obtaining a formulation containing anti-α4β7 antibody in high yield from a liquid solution containing the above, The method involves mixing-mode chromatography of a liquid solution containing an anti-α4β7 antibody and one or more impurities. By bringing the resin into contact with the aforementioned material, the anti-α4β7 antibody is bound to the resin, and The mixed mode chromatography resin is washed with the washing solution, and the pH is 4.2 The resin is brought into contact with an elution solution having the following pH and conductivity of 28 mS / cm or less. By eluting the anti-α4β7 antibody from the mixed-mode chromatography resin, This includes obtaining a formulation containing an anti-α4β7 antibody in high yield.

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

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

[0032] In some embodiments, the elution solution is NaCl with a concentration of approximately 160 mM to approximately 240 mM. Includes.

[0033] In some embodiments of the above-described model, the mixed-mode chromatography resin is resin 1 At least 55 g of anti-α4β7 antibody is brought into contact with L. In certain embodiments, The combined mode chromatography resin contains approximately 55g to 80g of anti-α4β7 inhibitors per liter of resin. They are made to make physical contact.

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

[0035] In some embodiments of the above-described model, the method uses a cation exchange (CEX) resin. The method further includes purifying the anti-α4β7 antibody. In some such embodiments, the CEX tree Lipids are manipulated in binding / elution mode.

[0036] In another aspect, the present invention relates to a liquid solution containing an anti-α4β7 antibody and one or more impurities from which an anti- A method for producing a preparation containing an α4β7 antibody, comprising an anti-α4β7 antibody and one or more By contacting a liquid solution containing impurities with a cation exchange (CEX) resin, anti-α4β 7. Binding the antibody to the resin, washing the CEX resin with a washing solution, and 16 mS / By bringing the resin into contact with an eluting solution having conductivity of less than 1 cm, anti-α4 is removed from the CEX resin. By eluting the β7 antibody, a formulation containing purified anti-α4β7 antibody is obtained, The anti-α4β7 antibody has a heavy chain variable region as described in SEQ ID NO: 1 and a light chain variable region as described in SEQ ID NO: 2. The method is characterized by including the region.

[0037] In one embodiment of the above-described model, the method comprises a liquid containing an anti-α4β7 antibody and one or more impurities. This is for obtaining a formulation containing reduced levels of HMW aggregates from a body solution, and the aforementioned method The method involves bringing a liquid solution containing an anti-α4β7 antibody and one or more impurities into contact with the CEX resin. By doing so, the anti-α4β7 antibody is bound to the resin, and the CEX resin is used in the washing solution. Further cleaning and contact of the resin with an eluting solution having an conductivity of 16 mS / cm or less By doing so, the anti-α4β7 antibody is eluted from the CEX resin, thereby reducing the amount The present invention provides a formulation containing Bell's HMW aggregates.

[0038] In some embodiments of the above-described model, the eluting solution has an electrical conductivity of 14 mS / cm or less. In other embodiments, the elution solution has a conductivity of approximately 11-16 mS / cm. In other embodiments, the elution solution has an electrical conductivity of about 12-14 mS / cm.

[0039] In some embodiments of the above-described model, the elution solution has a concentration of approximately 70 mM to approximately 110 mM. It contains NaCl.

[0040] In some embodiments of the above-described model, the elution solution has a pH of approximately pH 5 to approximately pH 6. In certain embodiments, the elution solution has a pH of approximately 5.1 to 5.8.

[0041] In some embodiments of the above-described model, the anti-α4β7 antibody is present in approximately 25-7 units per liter of resin. The antibody is loaded into the CEX resin at a concentration of 0 g. In certain embodiments, the anti-α4β7 antibody is The antibody is loaded into the CEX resin at a concentration of approximately 30-60 g per liter of resin.

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

[0043] In some embodiments of the above-described model, the method involves a mixed-mode chromatography resin The method further includes purifying the anti-α4β7 antibody using the mixture. In some such embodiments, the mixture The binding mode chromatography resin is operated in binding / elution mode.

[0044] In another embodiment, the present invention relates to the major isoform of an anti-α4β7 antibody and one or more basic A method for producing a formulation containing an anti-α4β7 antibody from a liquid solution containing isoform species. The liquid solution containing an anti-α4β7 antibody and one or more basic isoform species is catalyzed. The anti-α4β7 antibody is bound to the resin by contacting it with a resin exchange (CEX) resin. Furthermore, the CEX resin is washed with a cleaning solution, and the elution has an electrical conductivity of 11 mS / cm or higher. By bringing the solution into contact with the resin, anti-α4β7 antibodies can be eluted from the CEX resin. The objective is to obtain a formulation containing purified anti-α4β7 antibody, and that the anti-α4β7 antibody corresponds to SEQ ID NO: 1 The method is characterized by including the heavy chain variable region described in [reference] and the light chain variable region described in SEQ ID NO: 2. .

[0045] In one embodiment of the above-described model, the method involves a major isoform of an anti-α4β7 antibody and one The reduced levels of anti-α4β7 antibody from a liquid solution containing the above basic isoform species This method is for obtaining a formulation containing a basic isoform species, and the method is for obtaining an anti-α4β7 anti A liquid solution containing the body and one or more basic isoform species is brought into contact with the CEX resin. This process involves binding the anti-α4β7 antibody to the resin and using the CEX resin in a washing solution. The process involves washing and then bringing the resin into contact with an eluting solution having an conductivity of 11 mS / cm or higher. By doing so, the anti-α4β7 antibody is eluted from the CEX resin, thereby reducing the level The invention provides a formulation containing a basic isoform species of the compound.

[0046] In one embodiment, the purified composition contains about 4% to about 20% basic isoforms. nothing.

[0047] In some embodiments of the above-described model, the eluting solution has a conductivity of 12 mS / cm or more. In other embodiments, the elution solution has a conductivity of approximately 11-16 mS / cm. In other embodiments, the elution solution has an electrical conductivity of about 12-14 mS / cm.

[0048] In some embodiments of the above-described model, the elution solution has a pH of approximately pH 5 to approximately pH 6. In certain embodiments, the elution solution has a pH of approximately 5.1 to 5.8.

[0049] In some embodiments of the above-described model, the anti-α4β7 antibody is present in approximately 25-7 units per liter of resin. The antibody is loaded into the CEX resin at a concentration of 0 g. In certain embodiments, the anti-α4β7 antibody is The antibody is loaded into the CEX resin at a concentration of approximately 30-60 g per liter of resin.

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

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

[0052] In some embodiments of the above-described model, the method involves a mixed-mode chromatography resin The method further includes purifying the anti-α4β7 antibody using the mixture. In some such embodiments, the mixture The binding mode chromatography resin is operated in binding / elution mode.

[0053] In any one embodiment of the above-described model, the antibody is derived from Chinese hamster ovaries (CHO ) These are produced within the host cell.

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

[0055] In any one embodiment of the above-described model, the anti-α4β7 antibody is a heavy chain antibody as described in SEQ ID NO: 1. It includes a variable region sequence and the light chain variable region sequence described in Sequence ID No. 5.

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

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

[0058] 1. Less than 1% HMW aggregation from a liquid solution containing anti-α4β7 antibody and one or more impurities. A method for obtaining a composition containing a body, The liquid solution containing the anti-α4β7 antibody and the one or more impurities contains protein A. By bringing the matrix into contact with the anti-α4β7 antibody, the anti-α4β7 antibody is bound to the protein A. To make, Washing the matrix containing protein A with a washing solution, By contacting the matrix with an elution solution with a pH of 3.2 to 4, the protein The anti-α4β7 antibody is eluted from the matrix containing A, and HMW aggregates of less than 1% are obtained. To obtain a composition containing, The aforementioned anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and is described in Sequence ID No. 4. The CDR3 domain, the CDR2 domain described in Sequence ID No. 3, and the domain described in Sequence ID No. 2 It includes a heavy chain variable region containing the CDR1 domain, and the CDR3 domain described in Sequence ID No. 8. In, the CDR2 domain described in Sequence ID No. 7, and the CDR1 domain described in Sequence ID No. 6 The method comprising a light chain variable region including a domain.

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

[0060] 3. The solid phase comprises one or more of the following: beads, gels, and resins, as described in item 2. method.

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

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

[0063] 6. The pH of the elution solution is 3.2 to 3.7, as indicated in one of items 1 to 5. Method of loading.

[0064] 7. From a liquid solution containing anti-α4β7 antibody and one or more impurities, the anti-α4β7 antibody and 0 A method for obtaining a composition containing less than 0.6% HMW aggregates, A solution containing an anti-α4β7 antibody and at least one impurity was subjected to hydrophobic interaction chromatography. Raffy (HIC) resin and the flow-through of the anti-α4β7 antibody through the HIC resin By contacting under conditions that enable this, the anti-α4β7 antibody and less than 0.6% H This includes obtaining a composition containing MW aggregates. The aforementioned HIC resin is characterized as a highly hydrophobic resin. The aforementioned anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and is described in Sequence ID No. 4. The CDR3 domain, the CDR2 domain described in Sequence ID No. 3, and the domain described in Sequence ID No. 2 It includes a heavy chain variable region containing the CDR1 domain, and the CDR3 domain described in Sequence ID No. 8. In, the CDR2 domain described in Sequence ID No. 7, and the CDR1 domain described in Sequence ID No. 6 The method comprising a light chain variable region including a domain.

[0065] 8. The HIC resin is equilibrated with a phosphate buffer with a pH of less than approximately 7.2. The method described in item 7.

[0066] 9. The phosphate buffer contains approximately 0.35 mM to approximately 0.15 mM potassium phosphate. The method described in item 8, including the method described in item 8.

[0067] 10. The resin load is approximately 55-75 mg / ml, according to any of items 7-9. One method.

[0068] 11. The composition contains less than approximately 0.22 ppm of residual protein A, item 7-1 A method that is one of 0.

[0069] 12. The composition contains less than 0.3 ppm of host cell protein (HCP), A method described in any one of items 7-11.

[0070] 13. The aforementioned highly hydrophobic HIC resin has an average pore size of approximately 50 to 150 μm, item 7 The method described in any one of the following 12 ways.

[0071] 14. The above-mentioned highly hydrophobic HIC resin has an average pore size of approximately 100 nm and / or approximately 100 μm A method according to any one of items 7 to 12, having a pore diameter of m.

[0072] 15. The antibodies described above were produced in Chinese hamster ovary (CHO) cells. The method described in any one of items 1 through 14.

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

[0074] 17. The anti-α4β7 antibody contains the heavy chain variable region sequence described in SEQ ID NO: 1 and SEQ ID NO: 5 A method according to any one of items 1 to 16, comprising the light chain variable region sequence described above.

[0075] 18. The anti-α4β7 antibody is vedolizumab, according to any one of items 1-16. Method of description. [Brief explanation of the drawing]

[0076] [Figure 1] The aggregates are shown as a function of the pH of the protein A eluate. [Figure 2-1] This plot shows the results of a predictive profiler assay screened for performance. [Figure 2-2] This plot shows the results of a predictive profiler assay screened for performance. [Figure 2-3] This plot shows the results of a predictive profiler assay screened for performance. [Figure 3]This graph compares vedolizumab with three other IgG antibodies and shows the pH characteristics for eluting each antibody from a cation exchange column. [Figure 4] This is a surface plot of a linear regression model showing the process recovery rate of Capto Adhere ImpRes against the pH and conductivity of the elution buffer. [Figure 5] This is a surface plot of a linear regression model showing the process recovery rate of Capto Adhere ImpRes against the pH and load volume of the elution buffer. [Figure 6] This is a surface plot of a linear regression model showing the percentage of HMW species of Capto Adhere ImpRes relative to the pH and conductivity of the elution buffer. [Figure 7] This is a surface plot of a linear regression model showing the percentage of HMW species of Capto Adhere ImpRes relative to the pH and load volume of the elution buffer. [Figure 8] This is a surface plot of HMW showing the effect of the pH of the elution buffer and the conductivity of the elution buffer on the percentage of HMW species (%). [Figure 9] This is a surface plot of HMW clearance showing the effects of the pH and conductivity of the elution buffer on HMW clearance. [Figure 10] This is a surface plot of monomers showing the effect of the pH of the elution buffer and the conductivity of the elution buffer on the percentage of monomer species (%). [Figure 11] This is an acidic surface plot showing the effect of the pH and conductivity of the elution buffer on the percentage of acidic isoform species. [Figure 12] This is a major surface plot showing the effect of the pH and conductivity of the elution buffer on the percentage of major isoform species. [Figure 13] This is a basic surface plot showing the effect of the pH and conductivity of the elution buffer on the percentage of basic isoform species (%). [Modes for carrying out the invention]

[0077] This invention relates to an anti-α4β7 antibody or its antigen-binding fragment, such as vedolizumab, purified Product-related substances present in the formulation (e.g., aggregates such as high molecular weight (HMW) aggregates, etc.) Sfolded chemical species or protein fragments) and / or process-related impurities (e.g.) Examples include host cell proteins (HCPs), host cell nucleic acids, viruses, and chromatography materials. The present invention relates particularly to a purification method for controlling the amount of a substance (and / or culture medium component).

[0078] I. Definition To make the present invention easier to understand, we will first define certain terms.

[0079] The cell surface molecule "α4β7 integrin" or "α4β7" (the entire specification) (Used interchangeably throughout) α4 chain (CD49D, ITGA4) and β7 chain (ITGB 7) is a heterodimer with the human α4 integrin and β7 integrin genes (and That is, GenBank (National Center for Biotechno (Logy Information, Bethesda, Md.) RefSeq Access (Sequence numbers NM_000885 and NM_000889) are B and T lymphocytes, especially me It is expressed by Molly CD4+ lymphocytes. A typical characteristic of many integrins is that, α4β7 can exist in a resting or activated state. Ligands for α4β7 include vascular cells. Cellular adhesion molecules (VCAM), fibronectin and mucosal adrenin (MAdCAM (for example) Examples include MAdCAM-1). Antibodies that bind to α4β7 integrin are described in this specification. In textbooks, it is called "anti-α4β7 antibody."

[0080] The antibodies or anti- The somatic conjugate fragment binds to α4β7, but also to α4β1. E It does not bind to β7 either. Mab is an example of an antibody that exhibits binding specificity to the α4β7 complex.

[0081] As used herein, the term "antibody" refers to molecules linked together by disulfide bonds. Immunoglobulin components consist of four polypeptide chains: two heavy (H) chains and two light (L) chains. This refers to the child. Each heavy chain has a heavy chain variable region (abbreviated as HCVR or VH in this specification). It consists of a heavy chain steady region and a heavy chain steady region. The heavy chain steady region consists of three CH1, CH2, and CH3. It consists of domains. Each light chain has a light chain variable region (LCVR or VL in this specification). It consists of a (abbreviated as) and a light chain steady region. The light chain steady region consists of one domain CL. The VH and VL regions are separated by a more conserved region called the Framework Region (FR). It can be further divided into hypervariable regions called complementarity-determining regions (CDRs). VH and VL are FR1, CDR1, FR2, and CD, arranged from the amino terminus to the carboxyl terminus. It consists of three CDRs and four FRs arranged in the order R2, FR3, CDR3, and FR4. In some embodiments, the antibody is a "crystallizable fragment". It has an anti-crystallizable (Fc) region. In certain embodiments, The body is of the IgG1 isotype and possesses a κ light chain.

[0082] "CDR" or "Complementarity Determination Area" is more commonly referred to as a framework area (FR). It is a region of hypervariability intervening within a conserved region.

[0083] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” of an antibody are, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies, functional heavy chain antibodies (nanobo D), and at least one antibody that competes with intact antibodies for specific binding. This refers to any part of an antibody that has specificity for a desired epitope (for example, epitope). A complementarity-determining region having a framework sequence sufficient to specifically bind to the target was isolated. (The part that was formed). The antigen-binding fragment was obtained by recombinant technology, or by enzymatic or chemical cleavage of the antibody. It can be manufactured by [this method].

[0084] The "humanized" form of non-human (e.g., rodent) antibodies refers to the minimal amount of non-human antibodies derived from them. It is a chimeric antibody containing a sequence. In many cases, humanized antibodies are derived from the hypervariable region of the recipient. The residues possess the desired specificity, affinity, and functionality in mice, rats, rabbits, and... Alternatively, the residue may be substituted with a residue derived from the hypervariable region (donor antibody) of a non-human species such as a non-human primate. It is a human immunoglobulin (recipient antibody). In some cases, the human immunoantibody Framework region (FR) residues are replaced with corresponding non-human residues. Furthermore, humanized antibodies... These may include residues not found in either the recipient antibody or the donor antibody. Modification is performed to further improve the performance of the antibody. Generally, humanized antibodies are hypervariable CD. All or almost all of the R loop corresponds to that of a non-human antibody, and all or most of the FR corresponds to that of a non-human antibody. At least one, typically two, variable sequences, all of which are human antibody sequences. It includes almost all of the main components. Humanized antibodies may, in some cases, have a smaller antibody constant region (Fc). This also includes, in part, human antibodies. For further details, see, for example, Jones et al., Nature 321:522-525 (1986), Ri echmann et al., Nature 332:323-329 (1988), and Presta, Curr. Struct. Biol. 2: 593-596 (1992) Please refer to ).

[0085] As used herein, "recombinant antibody" refers to an antibody that is introduced into a host cell, such as a mammalian cell. Transcription and translation of genes(s) incorporated into the recombinant expression vector(s)(s) This refers to the antibody produced as a result of [the process]. In certain embodiments, the recombinant protein is IgG. (For example, IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, An isotype antibody selected from the group consisting of IgD or IgE. Specific implementation Morphologically, the recombinant antibody is IgG1.

[0086] The term "recombinant host cell" (in this specification, it is used interchangeably with the term "host cell") This includes cells into which recombinant expression vectors have been introduced. Such terminology is used for specific subjects. It is understood that the term is intended to refer not only to cells but also to the offspring of such cells. It should be so. Because certain modifications can occur in subsequent generations due to mutation or environmental influences. Although such offspring may not actually be identical to the parent cell, as used herein. This falls within the scope of the term "host cell." Furthermore, unless otherwise specified, for example, The term "cell" is used in phrases such as chief cell, mammalian cell, or mammalian host cell. If so, it should be understood that this is intended to include a population of cells.

[0087] As used herein, the term "vector" refers to a vector that amplifies another nucleic acid to which it is linked. This refers to nucleic acid molecules that can replicate. This term includes vectors as self-replicating nucleic acid structures. —and vectors incorporated into the genome of the introduced host cell. Specific vectors - The vector can induce the expression of nucleic acids to which it is functionally linked. — is referred to as “expression vector” in this specification.

[0088] In this specification, the term "upstream process" as used in relation to proteins refers to the host This refers to the activity of producing and recovering proteins (e.g., antibodies) from cells (e.g., antibodies). (For example, in cell culture to produce target proteins.)

[0089] As used herein, the term "downstream process" refers to, for example, a protein such as the antibody of interest. This refers to one or more methods used after the upstream process for purifying the substance. For example, downstream Process methods include, for example, protein A affinity chromatography. Affinity chromatography, anion or cation exchange ion exchange chromatography Ion exchange chromatography like Graphy, size exclusion chromatography, mixed Intermodal chromatography, hydrophobic interaction chromatography (HIC), or One example is the purification of protein products using conversion chromatography.

[0090] As used herein, the terms "culture" and "cell culture" refer to cells being cultured under controlled conditions. Generally, this refers to the process of growing cells outside of their natural environment. "To" means to keep cells in a condition suitable for cell survival and / or growth and / or proliferation. This refers to contact with a culture medium. In certain embodiments, cell culture refers to the process of obtaining the recombinant protein of the target. To generate and maintain a population of host cells capable of producing, for example, anti-α4β7 antibodies. This refers to methods for producing and recovering target proteins, as well as methods and techniques for producing and recovering target proteins. For example, after an expression vector is incorporated into a suitable host, such as a host cell in culture, The main factors are the expression of the relevant nucleotide coding sequences and the desired recombinant protein. It can be maintained under conditions suitable for recovery and purification. "Cell culture" is a solution containing cells. It can also refer to a liquid.

[0091] As used herein, "clarified recovery" refers to the removal of cellular debris and particulate impurities from the material. Cell culture, for example, after one or more steps have been taken to remove solid particles such as material. A liquid containing the target protein extracted from a yeast bioreactor, such as an anti-α4β7 antibody. This refers to the material. After cell culture, the recovered material is usually purified using separation techniques such as centrifugation and filtration. Cells and cell debris are removed. The initial clarification and particle removal process, for example, "Clarified cells" that can be used in subsequent chromatography steps (downstream processes) The collected material is obtained. The clarified recovered material is generally used in the downstream process described herein. It is a starting material in downstream processes such as Seth.

[0092] As used herein, "chromatographic support" refers to a specific chemical composition or specific Having a three-dimensional structure, or affinity chromatography, gel filtration (size Chromatography including exclusion chromatography or ion exchange chromatography. A solid or polycrystalline material can be used to immobilize specific chemical groups or macromolecules in order to perform the operation. This refers to a porous matrix. Examples of chromatography supports are limited to these. Examples include resins (e.g., agarose) or films, although these are not used in this specification. The "chromatography housing" used is a housing that contains the chromatography support. It refers to a structure that contains a column or cartridge. Examples of chromatography housings include columns or cartridges. Examples include a jar or other container.

[0093] As used herein, "buffer" refers to the action of its acid-base conjugate component. It refers to an aqueous solution that is more resistant to changes in pH. "Buffer" refers to a process step or chromatography. Specific conditions for controlling chromatography supports such as tography resins or membranes Used to establish a set.

[0094] As used herein, the term “equilibrium solution” refers to a process step or chromatography Support, for example, formulated to establish initial operating conditions for a chromatography operation. It refers to an aqueous solution. An equilibration solution is used to load the target protein, for example, an antibody. Used to prepare solid phases, such as chromatography supports, resins, or films. ru.

[0095] As used herein, the terms “cleaning solution” or “cleaning liquid” refer to resins or films, etc. Aqueous solution formulated to replace unbound contaminants from the chromatographic support. This refers to... In some embodiments, the washing solution loads the target protein, for example, an antibody... Afterwards, and before eluting the target protein, such as an antibody, a solid support, such as a resin, is applied. Or it is passed through a membrane. In one embodiment, the washing solution has biochemical properties similar to those of the equilibration solution. ru.

[0096] As used herein, "flow-through operation" refers to the process of removing proteins from the matrix during washing. For example, it does not substantially bind to and / or elutes hydrophobic chromatography resins. In contrast, processes that leave impurities bound to the chromatography support To point.

[0097] As used herein, the terms “elution solution” or “eluting solution” refer to chromatography. A support, such as a resin or film, is formulated to replace the target protein, such as an antibody. This refers to an aqueous solution in which the target protein, such as an antibody, is extracted. Lammagraphy support, for example, a resin or film, is designed to associate more readily with the elution solution than other materials. It has different biochemical properties from the balancing solution and / or washing solution.

[0098] The term "impurities" used herein refers to impurities present in a solution containing the antibody to be purified. The term "impurity" includes both process-related impurities and product-related impurities.

[0099] As used herein, the term "process-related impurities" refers to compositions containing proteins. For example, impurities that are present in the solution but do not originate from the protein itself (or multiple impurities) This refers to impurities. For example, process-related impurities are not limited to these. However, cell culture medium components, host cell components (e.g., proteins (HCP), host cell nucleic acids, and This includes lipid-containing intracellular structures or fragments thereof, viruses, trace metals from buffers, and Examples include leachings from ions, material handling containers, or chromatography supports. Process-related impurities can be found in proteins, such as antibodies, during preparation (upstream and / or downstream). It can be generated during a flow process.

[0100] As used herein, the term “host cell impurities” refers to host cell lines, cell culture media, and This refers to any proteinaceous, nucleic acid, lipid, or other contaminants introduced through cell culture. This refers to the product. Examples of impurities are not limited to these, but include Chinese Hamster ovary protein (CHOP), E. coli protein, enzyme protein, monkey CO S protein, or myeloma cell protein (e.g., NS0 protein (BALB / c) Examples include mouse plasmacytomas derived from mice.

[0101] As used herein, the term "product-related impurities" refers to the target protein, for example, The product contains impurities originating from the antibody itself. For example, product-related impurities are not limited to these. It is not something that can be formed, but aggregates (e.g., HMW), misfolded chemical species, oxidation, Examples include deamidated chemical species or low molecular weight fragments of the antibody of interest.

[0102] As used herein, the terms "aggregates" or "plural aggregates" refer to multiple antibodies. This refers to the binding of antibody fragments. For example, aggregates are dimers of antibodies and / or antibody fragments, and tri It may be a mer, a tetramer, or a polymer larger than a tetramer. Antibody aggregates are soluble or It can be insoluble. The bonds between aggregated molecules are covalent, regardless of the mechanism by which the molecules bond. Or it may be non-covalent. The bond may be direct between aggregated molecules, or This can also be indirect, involving other molecules that link molecules together. An example of the latter is... However, these are not the only examples, but also include disulfide bonds with other proteins and lipids. Hydrophobic bonding with DNA, charge bonding with DNA, affinity bonding with leached protein A, Alternatively, mixed mode binding with multiple components may occur. Aggregates are proteins in cell culture. Irreversibly formed during expression, during downstream protein purification processes, or during drug storage. It is possible. The presence of aggregates in solution can be detected, for example, by size exclusion chromatography (SEC). (For example, SEC by UV detection, SEC by light scattering detection (SEC-LSD)), Gold flow fractionation, sedimentation velocity method in ultracentrifugation analysis, or capillary electrolysis Electrophoresis - Determination is performed using sodium dodecyl sulfate (CE-SDS, reduced and unreduced forms). It is possible.

[0103] The term "high molecular weight" or "HMW" refers to antibodies with a molecular weight larger than that of monomer antibodies. It is used to refer to antibody complexes. In one embodiment, the HMW aggregates are approximately 147 kDa They also have large molecular weights. The presence of high molecular weight aggregates can be observed, for example, in size exclusion chromatography. It can be determined by standard military tactics well known in the relevant technical field, such as SEC. ru.

[0104] "Substantially purified" with respect to the desired protein means purified protein containing The sample 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%, and 97.5%, at least 98%, at least 98.5%, or at least 99% Contains desired recombinant protein, with impurities of less than 3%, less than 2.5%, less than 2%, and 1.5%. This means full, less than 1%, or less than 0.5%.

[0105] The term "approximately" indicates that the value that follows is not an exact value, but within a range of ±5% of that value. This means it is the center point. If that value is a relative value given as a percentage. The term "approximately" indicates that the value that follows is not an exact value, but within a range of ±5% of that value. This is the center point, and it means that the upper limit of the range cannot exceed a value of 100%.

[0106] II. Methods and Compositions Related to Antibody Purification This specification describes how to obtain a solution from, for example, a liquid solution, or from, for example, a clarified product of a mammalian cell culture. A method for purifying anti-α4β7 antibodies such as dolizumab is provided. The present invention provides a method for purifying antibody solutions Examples of substances present in the liquid include cell culture medium components, host cell proteins (HCPs), and host cell nucleic acids. , viruses, and process-related impurities such as chromatography materials, as well as aggregates ( (Including HMW aggregates), misfolded chemical species, or fragments of the target protein, etc. In certain aspects of antibody purification processes that reduce the level of impurities, including product-related impurities, This is at least partially based on the present invention. The method of the present invention is based on an anti-α4β7 antibody, particularly Vedolizuma Purify antibodies that have a binding region (i.e., CDR or variable region) for vedolizumab or vedolizumab. This is useful in that it allows for the formulation of antibodies for use in human patients. can.

[0107] More specifically, the methods disclosed herein are for low levels of antibody aggregation, such as HMW antibody aggregation. It is useful in obtaining the body. In certain embodiments, the method disclosed herein is about 0% ~5.0% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%) 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1. 5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3% , 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3 0.2%, 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 The present invention provides compositions having aggregates of %, 4.9%, or 5%, such as HMW aggregates. In certain embodiments, the methods disclosed herein involve about 0% to 2%, 2% or less, and 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 Aggregates containing 0.7% or less, 0.6% or less, or 0.5% or less, for example, HMW aggregates. The present invention provides a composition comprising an anti-α4β7 antibody and the low-level HMW aggregates. Compositions are also included. More specifically, the anti-α4β7 antibody vedolizumab, or Antibodies containing the antigen-binding domain of vedolizumab can be produced. Vedolizumab also Vedolizumab is known by the brand name ENTYVIO (registered trademark) (Takeda Pharmaceutical Company). This involves the framework region and constant region of human IgG1, and the antibody derived from the mouse antibody Act-1. This is a humanized antibody containing the original conjugated CDR. It includes the CDR, variable region, and mutation F of vedolizumab. The c region (which has been mutated to eliminate the Fc effector function) is referenced in this book. It is described in U.S. Patent No. 7,147,851, which is incorporated in full in the specification.

[0108] Vedolizumab specifically binds to α4β7 integrins, such as the α4β7 complex, and α Interaction between 4β7 integrin and mucosal adresin cell adhesion molecule 1 (MAdCAM-1) This blocks the process, preventing memory T lymphocytes from passing through the endothelium and migrating into the inflamed gastrointestinal parenchymal tissue. Vedolizumab is a humanized monoclonal antibody that inhibits α4β1 and αEβ7. It does not bind to integrins, nor does it inhibit their function, and does not bind to α4 integrins or vascular cell adhesion molecules. The interaction with 1 (VCAM-1) is not antagonistized.

[0109] α4β7 integrin is a specific sub-sub It is expressed on the surface of the set. MAdCAM-1 is mainly expressed in intestinal endothelial cells and T phosphorus α4β7 integrin plays an important role in the homing of pacyl cells to intestinal lymphoid tissue. The interaction between MAdCAM-1 and chronic inflammation, which is a characteristic of ulcerative colitis and Crohn's disease. Vedolizumab has been shown to be an important contributing factor to mucosal inflammation. inflammatory bowel diseases including Crohn's disease and ulcerative colitis, pouchitis including chronic pouchitis, transplantation It can treat universal host disease and HIV.

[0110] The heavy chain variable region of vedolizumab is provided herein as Sequence ID No. 1, and vedolizumab The light chain variable region of vedolizumab is provided herein as Sequence ID No. 5. Heavy chain variable region including CDR1 of number 2, CDR2 of sequence number 3, and CDR3 of sequence number 4. Includes. Vedolizumab is CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and SEQ ID NO: It includes a light chain variable region containing CDR3 of 8. In one embodiment, the antibody is amino of SEQ ID NO: 9 It comprises a heavy chain containing an acid sequence and a light chain containing the amino acid sequence of SEQ ID NO: 10. Vedolizumab and The sequences of vedolizumab are all incorporated herein by reference in their entirety. U.S. Patent Application Publication No. 2014 / 0341885 and U.S. Patent Application Publication No. 2014-03 It is also described in Patent No. 77251. The methods disclosed herein are as described above and in the attached sequence listings. This procedure is performed using an antibody that contains a binding region (e.g., CDR or variable region) as described. It is possible.

[0111] Methods for producing antibodies are well known in the relevant field. Mammalian hosts are anti-α4β The system is engineered to stably express 7 antibodies (e.g., vedolizumab). For the overall cell culture process and considerations in the production of any monoclonal antibody, please refer to [reference]. Li et al. (2010) mAbs 2:5,466 is incorporated herein by reference. -477, and Birch and Racher (2006) Adv. Drug De This is described in livery Rev.58:671-685.

[0112] When using cell culture methods, anti-α4β7 antibodies may be produced intracellularly, or circumcellularly. It may be produced in the marginal space or secreted directly into the culture medium. Anti-α4β7 antibody is released into the cells In embodiments produced within, host cells or lysed cells (e.g., homogenizer) Particulate fragments (generated by the suction) are subjected to centrifugation, although not limited to these. Alternatively, it can be removed by various means, including filtration. Anti-α4β7 antibody in the culture medium If secreted, the supernatant from such an expression system is taken using a commercially available protein concentration filter. First, concentrate it.

[0113] Add one or more processes to the culture medium or lysate, such as precipitation, flocculation, centrifugation, and / or filtration. The cell culture supernatant, or clarified supernatant, is obtained by removing particulate cell debris through a processing step. It forms a receptacle. Then, an antibody, such as an anti-α4β7 antibody (e.g., vedolizumab or vedolizumab), is added. The antibody (which has a binding region equivalent to dolizumab) is purified as described in detail below. impurities (e.g., cell culture medium components, host cell proteins (HCPs), host cell nucleic acids, etc.) Process-related impurities such as russ and chromatography materials, as well as aggregates (HMW Formation of aggregates, misfolded chemical species, or fragments of the target protein. Removes (material-related impurities).

[0114] The purification process uses the upstream manufacturing method described above and / or another conventional manufacturing method. Therefore, it can be started after the antibody has been manufactured. A clarified solution or mixture containing the antibody. After the compound is obtained, process-related impurities such as other proteins produced by the cells are removed. Furthermore, the antibody of the target is separated from product-related impurities. In certain non-limiting embodiments, Such separations are performed using CEX, AEX, and / or MM chromatography. In certain embodiments, the process may include affinity separation steps and ion exchange separation steps. (Multiple), mixed-mode steps (multiple), and / or hydrophobic interaction separation steps (multiple) A combination of one or more different purification methods, including ), may also be used. The purification process involves separating the antibody mixture based on the antibody's charge, hydrophobicity, and / or size. The goal is to separate. In one aspect of the present invention, such further separation steps involve hydrophobic and anionic chromatography involving sex, or cationic, interactions (or a combination thereof). This is done using [a specific method]. Many chromatography resins are used for each of these methods. It is available for sale, and the purification scheme can be precisely adjusted to suit the specific antibody involved. Each of these separation methods involves flowing the antibody through a column at a different rate, and the antibody is then separated. This achieves increasing physical separation as the material passes further through the ram, or separation resins. It is then possible to selectively adhere to (or a medium). Then, using different eluents, The body is eluted differentially. In some cases, the target antibody is eluted because impurities are specifically eluted by the column resin. If the target antibody does not bind, it is separated from the impurities (i.e., the target antibody is f In other cases, the antibody of interest adheres to the resin of the column, along with impurities (which are contained in the raw-through solution). The product-related substances are eluted from the column resin during the washing cycle, and then the anti The body is released by changing the liquid surrounding the resin, and the target antibody is eluted from the column.

[0115] In a particular embodiment, in the "capture step," the antibody-containing solution is processed using affinity chromatography. Antibodies are purified from impurities using chromatography. In certain embodiments, chromatography is used. The material can selectively or specifically bind to the target antibody ("capture"). Non-limiting examples of such chromatography materials include protein A and protein G. For example, a chromatography material containing an antigen to which the target antibody binds, and an Fc-binding agent. Examples include chromatography materials containing protein.

[0116] In certain embodiments, the affinity chromatography steps described herein are The clarified recovered product containing the anti-α4β7 antibody is then processed into a protein A matrix (e.g., prote This includes subjecting the column to a chromatography column containing an A resin. In certain embodiments, this includes subjecting the column to a chromatography column containing an A resin. Protein A is involved with various antibody isotypes, particularly IgG1, IgG2, and IgG It is useful for affinity purification and isolation of protein 4. Protein A is mainly its Fc region. Protein A is a bacterial cell wall protein that binds to mammalian IgG via [a specific mechanism]. In its natural state, it possesses five IgG-binding domains and other domains of unknown function. do.

[0117] Purification of anti-α4β7 antibody using protein A resin In one embodiment, the method described herein involves using protein A to obtain an antibody and one or more Liquid solutions containing impurities, for example, anti-α4β7 antibodies from clarified recovered material (e.g., This method includes the purification of vedolizumab. This includes binding to a matrix for tea chromatography. In certain embodiments, Antibody solutions should have concentrations greater than 10 g / L, for example, 10-50 g / L, 20-45 g / L, or 30- It can be loaded onto the matrix for affinity chromatography at a concentration of 40 g / L. For example, antibody solutions are available in concentrations of approximately 10 g / L, 11 g / L, 12 g / L, and 13 g. / L, 14g / L, 15g / L, 16g / L, 17g / L, 18g / L, 19g / L, 2 0g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L , 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L, 39g / L, 4 0g / L, 41g / L, 42g / L, 43g / L, 44g / L, 45g / L, 46g / L Protein A affinity at 47g / L, 48g / L, 49g / L, or 50g / L - It can be loaded onto a chromatography matrix.

[0118] Protein A resin is sold by multiple vendors. One suitable resin is GE Heal There is MabSelect (trademark) sold by thcare. Suitable resins include: These are not the only examples, but also include Mab sold by GE Healthcare. Select SuRe(TM), MabSelect SuRe LX, MabSel ect, MabSelect Xtra, rProtein A Sepharose, MabSelect® ProA resin, sold by EMD Millipore. ProSep HC, ProSep Ultra, and ProSep Ultra Pl MabCapture, sold by Life Technologies, is one example. It is possible.

[0119] The Protein A column is equilibrated with a suitable equilibration solution prior to loading the sample. This can be done. After loading the column, the column is cleaned once with a suitable set of solutions. Washing multiple times can reduce one or more impurities, and the anti-α4β7 antibody is a proteo It remains connected to input A.

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

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

[0122] Next, the protein A column can be eluted using a suitable elution solution. For example... Alternatively, glycine-HCl, acetic acid, or citric acid can be used as the elution solution. In one embodiment, the elution solution is citric acid, for example, a sodium citrate elution solution. In some embodiments, the elution solution is about 3.0 to 4.0 (for example, about 3.1 to 4.0) 3.2~4.0, 3.3~4.0, 3.4~4.0, 3.5~4.0, 3.6~4.0, It can have a pH of 3.7-4.0, 3.8-4.0, or 3.9-4.0. In certain embodiments, the elution solution has a pH of approximately 3.0–3.4. Several embodiments In terms of morphology, the eluted solutions were approximately 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5. It can have a pH of approximately 3.6, 3.7, 3.8, 3.9, or 4.0. In some embodiments, the elution solution has a pH of 3.3 or higher. In some embodiments, the elution solution has a pH of 3.4 or higher. In some embodiments, the elution solution has a pH of 3.5 or higher. In some embodiments, the elution solution has a pH of 3.6 or higher. In the application form, the elution solution has a pH of 3.7 or higher. Several implementation forms In this state, the elution solution has a pH of 3.8 or higher. In some embodiments The elution solution has a pH of 3.9 or higher. Then it can be monitored using well-known methods. After recovering the target eluate fraction, further... It can be prepared to carry out the process.

[0123] As shown in the examples, the pH of the elution buffer for the protein A affinity column Compared to embodiments with a lower pH (e.g., 2.9-3.3), Protein A Affini Implementation with a higher pH (e.g., 3.3-4.0) of the elution buffer pH of the T column In this state, the eluate containing anti-α4β7 antibody has fewer impurities such as HMW aggregates. In that embodiment, the eluate contains anti-α4β7 antibody and is approximately 0% to 5.0% (for example, 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 Contains ~4.5%, or 0~5%, of HMW aggregates. In some embodiments, the eluate It contains anti-α4β7 antibody and is present in an amount of approximately 2% or less (for example, approximately 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 Lower, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less (Below: 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) It contains HMW aggregates. In certain embodiments, the eluate of the protein A resin contains anti-α4β7 Contains antibodies, and in concentrations of approximately 0% to 2%, 2% or less, 1.9% or less, 1.8% or less, and 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% Below, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% The following refers to aggregates with concentrations of 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less, for example. It contains HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 1.2 % or less of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 1.1% or less of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody, and contains about 1% or less (e.g., about 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 0.9 % or less of HMW aggregates.

[0124] The buffers and methods described herein can reduce the level of host cell protein (HCP) in compositions such as compositions containing an anti-α4β 7 antibody eluted from Protein A, compared to the level of HCP when an elution buffer without one or more of the parameters described herein is used. In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and less than about 250 ppm (e.g., about 240 ppm, 23 0 ppm, 220 ppm, 210 ppm, 200 ppm, 190 ppm, 180 ppm, 170 ppm, 160 ppm, 150 ppm, 140 ppm, 130 ppm, 120 ppm, 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 4 0 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, or less than 1 ppm) of HCP and contains a composition. In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and about 1 - 250 ppm (e.g., about 1 - 240 ppm, 1 - 230 ppm, 1 - 220 ppm). ppm). In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and about 1 - 250 ppm (e.g., about 1 - 240 ppm, 1 - 230 ppm, 1 - 220 ppm). ppm, 1~210ppm, 1~200ppm, 1~190ppm, 1~180ppm, 1~170ppm, 1~160ppm, 1~150ppm, 1~140ppm, 1~13 0ppm, 1~120ppm, 1~100ppm, 1~90ppm, 1~80ppm, 1 ~70ppm, 1~60ppm, 1~50ppm, 1~40ppm, 1~30ppm, 1 ~20ppm, 1~10ppm, 1~9ppm, 1~8ppm, 1~7ppm, 1~6p HCP (pm, 1-5 ppm, 1-4 ppm, 1-3 ppm, or 1-2 ppm) Includes compositions containing [specific components].

[0125] In one embodiment, an anti-α4β7 antibody bound to protein A is used in a pH higher than 3.3. (For example, pH 3.3-4.0, pH 3.4-4.0, pH 3.5-4.0, pH 3.6) Elution (~4.0, pH 3.7~4.0, pH 3.8~4.0, or pH 3.9~4.0) Elution with buffer reveals anti-α4β7 antibody and reduced levels of HMW aggregates. Elution and / or eluate containing anti-α4β7 antibody and / or reduced levels of HCP Obtained. In one embodiment, the elution solution has a pH of 3.3 to 3.9. The elution buffer has a pH of 3.3 to 3.8. In one embodiment, the elution buffer - The pH is 3.4 to 3.6. In one embodiment, the pH of the elution buffer is 3.4 It is ~4.0. In one embodiment, the elution buffer is citric acid, for example, sodium citrate. It contains, for example, 100 mM citric acid or 25 mM citric acid. In one embodiment, The Tein A affinity chromatography column contains 25 mM sodium citrate. Washed and eluted with a buffer, the washing buffers being 5.6-6.2, 5.7-5.9 Or the pH is 5.8, and the elution buffer is 3.3-3.9, 3.4-3.6 or The pH is 3.5.

[0126] In certain embodiments, the material loaded into the protein A resin is, for example, an anti-α4β7 anti- This is a clarified cell culture recovery obtained from recombinant cell lines expressing several different organisms. In this embodiment, the recombinant cell line (i.e., the host cell line) is a Chinese hamster ovary. (CHO) cells can be used. In some embodiments, CHO cells are glutamic GS-CHO cells can be created by deficient in the gene encoding synthase. In some embodiments, CHO cells have a gene encoding dihydrofolate reductase This can result in DHFR-CHO cells lacking the necessary properties.

[0127] The protein A eluate is adjusted for pH and / or conductivity for subsequent purification steps. This can be done. The protein A eluate is depleted prior to further chromatographic polishing steps. Filtration is performed through a filter to remove turbidity and / or various impurities from the target antibody. It may be removed.

[0128] Purification of anti-α4β7 antibodies using HIC Antibodies, for example, anti-α4β7 antibodies (e.g., vedolizumab or equivalent to vedolizumab) Antibodies having a binding region are described in detail below, and as described in Example 2, After purification with Rothein A, further purification can be performed using downstream process technology. The refining process, which takes place in the latter half of the process, is often called the "polishing" process, and the level of impurities is compared While the target level may be lower, considering the nature of antibodies intended for human use, an even lower level is possible. It presents unique challenges in terms of what is desirable.

[0129] In one embodiment, the method described herein is hydrophobic interaction chromatography (HI C) A liquid solution containing an antibody and one or more impurities using a resin, for example, a clarified solution This includes the purification of anti-α4β7 antibodies from the collected materials.

[0130] In one embodiment, the present invention reduces high molecular weight (HMW) aggregates from an anti-α4β7 antibody solution. A method comprising contacting an antibody solution with a hydrophobic interaction chromatography (HIC) resin. The present invention provides a method that includes causing [something]. Hydrophobic interaction chromatography (HIC) is Proteins and HIC resins (e.g., polymer matrices modified with hydrophobic ligands) By utilizing the reversible interaction with hydrophobic surfaces, based on the difference in their surface hydrophobicity Next, the protein is separated. Consider the hydrophobicity of anti-α4β7 antibodies such as vedolizumab. Furthermore, by using highly hydrophobic HIC resin in the purification process, HMW aggregates and residual proteins can be removed. It can remove α4A and / or host cell protein (HCP) contaminants, and is anti-α4 The β7 antibody flows through the HIC resin and does not bind. In some embodiments, as specified herein Highly hydrophobic HIC resins suitable for use in the manner described are available at Toyopearl H A compound like exyl-650C (Tosoh Biosciences) has a C6 group attached. It contains polymethacrylate-based materials.

[0131] In some embodiments, the HIC is used in "flow-through mode". As used herein, “flow-through fraction” refers to a fraction containing the resin provided herein. This refers to the proteins in the mobile phase buffer that are recovered in the fraction after passing through the column.

[0132] In some embodiments, a solution containing an anti-α4β7 antibody and at least one impurity is used. Hydrophobic interaction under conditions that enable flow-through of anti-α4β7 antibodies through HIC resin It is brought into contact with (HIC) resin. In one embodiment, the HIC resin has an average pore size of about 100 nm. and / or having a pore size of about 100 μm. In one embodiment, the HIC resin is about 7.2 μm It is equilibrated with a buffer having a pH of 5.5. In one embodiment, the HIC resin is approximately 5.5 It is equilibrated with a buffer having a pH of approximately 7.2. In one embodiment, the HIC resin is It is equilibrated with a buffer having a pH of approximately 5.5 to approximately 7. In one embodiment, the buffer This is a phosphate buffer. In one embodiment, the phosphate buffer is approximately 0.35 M Contains approximately 0.15M potassium phosphate. In one embodiment, the resin load is approximately 55-75 m It is g / ml.

[0133] In one embodiment, a method for purifying anti-α4β7 antibody using an HIC column is described as anti-α4β7 antibody This involves flowing the solution containing the substance through a column. In other words, purification is performed by flowing the solution through the column. This involves recovering the anti-α4β7 antibody contained within, with the contaminants remaining bound to the column, and the anti-α The 4β7 antibody and column were used at pH 5.2-6.5, 5.7-6.2, or approximately 5.9 for 15 minutes. Phosphates at concentrations of 0-300 mM, 175-250 mM, or approximately 200 mM, for example, It is in a solution containing potassium phosphate.

[0134] In some embodiments, such a method using a highly hydrophobic HIC resin is used to prevent α4 β7 antibody and approximately 0% to 2.0% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.0%). 5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3% 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, less than 1.9%, or less than 2% A composition can be obtained that includes HMW aggregates of ). In some embodiments, highly hydrophobic compositions can be obtained. Using a method employing a HIC resin, the anti-α4β7 antibody is mixed with approximately 2% or less (for example, approximately 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less Lower, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less Lower, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2 A composition can be obtained that contains HMW aggregates in a concentration of % or less, or 0.1% or less. In this embodiment, the method using a highly hydrophobic HIC resin is used to obtain an anti-α4β7 antibody and Approximately 0%~2%, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less Below, a composition containing HMW aggregates in amounts of 0.3% or less, 0.2% or less, or 0.1% or less. It can be obtained. In certain embodiments, such a method using a highly hydrophobic HIC resin is used. This allows you to obtain a composition containing an anti-α4β7 antibody and less than 0.6% HMW aggregates. In one embodiment, a composition containing an anti-α4β7 antibody and less than 0.5% HMW aggregates was obtained. In one embodiment, a composition comprising an anti-α4β7 antibody and less than 0.4% HMW aggregates is used. Furthermore, the composition contains less than approximately 0.3 ppm of host cell proteins (HCPs). It is possible for host cells to be Chinese hamster ovaries such as GS-CHO cells. CHO) cells were used. In one embodiment, the composition contained residual protein less than approximately 0.22 ppm. Includes nA.

[0135] Purification of anti-α4β7 antibody using mixed mode resin In one embodiment, this specification describes a mixed-mode chromatography resin as a binding / elution mode For use in a liquid solution containing antibodies and one or more impurities, such as clarified cell culture recovery. A method is provided for purifying an anti-α4β7 antibody, such as vedolizumab, from a substance. In that embodiment, the mixed-mode chromatography resin has a high impurity removal rate and high volume It has properties suitable for the quantity. In one embodiment, a mixing mode for purifying anti-α4β7 antibody. Chromatographic resins possess strong anion exchange, hydrogen bonding, and hydrophobic bonding properties. In another embodiment, a mixed-mode chromatography tree is used to purify the anti-α4β7 antibody. Lipids exhibit strong anion exchange on smaller beads, such as those with a diameter of approximately 35-45 μm. , possesses hydrogen bonding and hydrophobic bonding capabilities. In specific embodiments, as described herein The mixed-mode chromatography resin used in the method and composition is CAPTO® trademark. Adhere ImpRes(GE Healthcare Life Science (Currently Global Life Sciences Solutions, LLC) In certain embodiments, the mixed mo used in the methods and compositions described herein Adhere (GE Health) is a CAPTO™ trademarked chromatography resin. Care Life Sciences, now Global Life Sciences es Solutions, LLC) The clarified cell culture recovery is anti-α4β 7 Antibodies can be obtained from host cells that express antibodies by recombinant means. In certain embodiments, The chief cells are Chinese hamster cells such as GS-CHO cells or DHFR-CHO cells. - These can be ovarian (CHO) cells.

[0136] The mixed-mode chromatography method provided herein involves mixing an anti-α4β7 antibody into a mixed mode This includes, but is not limited to, bonding to a chromatographic resin. Affinity chromatography (e.g., protein A chromatography), Nione exchange (AEX) chromatography, cation exchange (CEX) chromatography , and further purification steps including hydrophobic interaction chromatography (HIC) are described in this specification. It can be used before and / or after the mixed-mode chromatography method described in this book. Therefore, in some embodiments, it is used in mixed-mode chromatography. The loading material is protein A eluate, AEX eluate, CEX eluate, or HIC eluate. The mixture provided herein may contain liquid or recovered HIC flow-through material. In some embodiments, the chromatographic method involves mixing a resin in a washing solution. This may further include washing and eluting antibodies from the resin.

[0137] In certain embodiments, at least 25 g / L (for example, at least 25 g / L, 30 g) / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 6 5g / L, 70g / L, 75g / L, 80g / L, 85g / L, 90g / L, 95g / L Load an antibody solution (or 100 g / L) onto a mixed-mode chromatography resin. This can be done. For example, mixed-mode chromatography of an antibody solution containing at least 55 g / L. It can be loaded into resin. In certain embodiments, the concentration is approximately 25 g / L to approximately 100 g / L. For example, approximately 25g / L to approximately 95g / L, approximately 25g / L to approximately 90g / L, approximately 25g / L to approximately 85g / L, approximately 25g / L to approximately 80g / L (for example, approximately 30g / L to approximately 80g / L, approximately 3 5g / L~Approx. 80g / L, Approx. 40g / L~Approx. 80g / L, Approx. 45g / L~Approx. 80g / L, Approx. 50g / L ~ Approx. 80g / L, Approx. 55g / L ~ Approx. 80g / L, Approx. 60g / L ~ Approx. 80g / L, approximately 65g / L to approximately 80g / L, approximately 70g / L to approximately 80g / L, or approximately 75g / L to An antibody solution of approximately 80 g / L can be loaded into a mixed-mode chromatography resin. For example, a mixed-mode chromatography tree can be used to analyze an antibody solution containing approximately 55 g / L to 80 g / L. It can be loaded into fat.

[0138] The resin should be washed, if necessary, with a suitable washing buffer that does not elute the antibodies bound to the resin. It can be cleaned. In one embodiment, the resin can be cleaned with sodium phosphate washing as needed. It can be washed with a buffer. In some embodiments, the washing buffer has a pH of 10 mM sodium phosphate, 25 mM sodium phosphate, neutral or near neutral (e.g., pH 6-8) Contains sodium phosphate, 50 mM sodium phosphate, or 75 mM sodium phosphate. It is possible. Other suitable washing buffers compatible with mixed-mode chromatography are also widely available. It is readily available.

[0139] This specification describes the preparation of anti-α4β7 antibodies after elution from mixed-mode chromatography resins. To increase the yield of anti-α4β7 antibody in the agent and / or the level of aggregates (e.g., HMW species ( This section describes a buffer that reduces (%). It increases the yield of anti-α4β7 antibody and / or Alternatively, to reduce the level of aggregates (e.g., HMW species (%)), a mixed-mode elution buff is used. The pH and / or conductivity of the ion can be adjusted. Mixed-mode chromatography Suitable elution solutions that match this are widely available. In some embodiments, a mixing mode The chromatography elution solution is a buffer such as citrate, acetate, or phosphate. Includes.

[0140] In some embodiments, mixed-mode chromatography is performed using the method described herein. The elution buffer for use with the resin should have a pH of 3.5 or higher (for example) pH 3.6 or higher, pH 3.7 or higher, pH 3.8 or It is higher than that, pH 3.9 or higher, pH 4.0 or higher pH 4.1 or higher, pH 4.2 or higher, pH 4.3 or It is higher than that, or pH 4.4 or higher, or pH 4.5 or higher. It has a higher value than this. For example, for use with mixed-mode chromatography resins. The elution buffer may have a pH of 3.9 or higher. In the embodiments, the mixed-mode chromatography resin is used in the manner described herein. The elution buffer for this purpose should have a pH of approximately 3.9 to 4.5 (for example, approximately 3.9 ~pH4.5, approx.pH3.9~pH4.4, approx.pH3.9~pH4.3, approx.pH3 pH 0.9 to approximately pH 4.2, approximately pH 3.9 to approximately pH 4.1, or approximately pH 3.9 to approximately pH 4.0 It has a pH of . In some embodiments, mixed-mode chromatography provided herein The elution buffer used in the fluoroscopy method should have a pH of approximately 3.9 to 4.4. It is possible to have.

[0141] In addition to or in alternative embodiments described above, mixing is performed in the manner described herein. The elution buffer for use with mode chromatography resins should be pH 4.5 or higher. A pH lower than that (for example, pH 4.4 or lower, pH 4.3 or lower) Lower than, pH 4.2 or lower, pH 4.1 or lower, pH 4 0.0 or lower, pH 3.9 or lower, pH 3.8 or lower Lower than, pH 3.7 or lower, pH 3.6 or lower, It has a pH of 3.5 or lower. For example, mixed-mode chromatography trees The elution buffer for use with lipids should have a pH of 4.2 or lower. This can be done. In certain embodiments, mixed-mode chromatography can be performed using the method described herein. The elution buffer for use with the ferrite resin should have a pH of approximately 4.2 to 3.5 (example). For example, approximately pH 4.2 to approximately pH 3.6, approximately pH 4.2 to approximately pH 3.7, approximately pH 4.2 to approximately pH H3.8, approximately pH 4.2 to approximately pH 3.9, approximately pH 4.2 to approximately pH 4.0, or approximately pH 4 It has a pH of 0.2 to approximately pH 4.1. For example, it can be used with mixed-mode chromatography resins. The elution buffer used can have a pH of approximately 4.2 to 3.8. ru.

[0142] In some embodiments, mixed-mode chromatography is used in the method described herein. The elution buffer for use with the resin should be approximately 40 mS / cm or less (for example, approximately 39 mS / cm). mS / cm, 38mS / cm, 37mS / cm, 36mS / cm, 35mS / cm, 34 mS / cm, 33mS / cm, 32mS / cm, 31mS / cm, 30mS / cm, 29 mS / cm, 28mS / cm, 27mS / cm, 26mS / cm, 25mS / cm, 24 mS / cm, 23mS / cm, 22mS / cm, 21mS / cm, 20mS / cm, 19 mS / cm, 18mS / cm, 17mS / cm, 16mS / cm, 15mS / cm, 14 mS / cm, 13mS / cm, 12mS / cm, 11mS / cm, or 10mS / cm It has the following conductivity. For example, for use with mixed-mode chromatography resins. The elution buffer can have a conductivity of approximately 30 mS / cm or less. Specific implementation In this state, the mixed-mode chromatography resin used in the method described herein The elution buffer used for this purpose is approximately 10 mS / cm to 40 mS / cm, for example, approximately 15 mS / cm. It has an electrical conductivity of approximately 35 mS / cm or approximately 20 mS / cm to 30 mS / cm. For example, the elution buffer for use with mixed-mode chromatography resins is approximately 20 It can have an electrical conductivity of approximately 30 mS / cm to about 30 mS / cm.

[0143] In addition to or in alternative embodiments described herein, the methods described herein may be used. The elution buffer for use with mixed-mode chromatography resins is approximately 30 mS / Less than or equal to (for example, 29 mS / cm, 28 mS / cm, 27 mS / cm, 26 mS / cm) , 25mS / cm, 24mS / cm, 23mS / cm, 22mS / cm, 21mS / cm , 20mS / cm, 19mS / cm, 18mS / cm, 17mS / cm, 16mS / cm , 15mS / cm, 14mS / cm, 13mS / cm, 12mS / cm, 11mS / cm It has a conductivity of , or 10 mS / cm or less. For example, mixed-mode chromatography - The elution buffer for use with the resin must have a conductivity of 28 mS / cm or less. Yes, it is possible. In certain embodiments, mixed-mode chromatography can be performed in the method described herein. The elution buffer for use with roughy resin should be approximately 10 mS / cm to 40 mS / cm. (For example, approximately 15 mS / cm to approximately 35 mS / cm, approximately 18 mS / cm to approximately 35 mS / cm) , approx. 11mS / cm ~ approx. 30mS / cm, approx. 12mS / cm ~ approx. 30mS / cm, approx. 13 mS / cm ~ approx. 30mS / cm, approx. 14mS / cm ~ approx. 30mS / cm, approx. 15mS / c m ~ approx. 30mS / cm, approx. 16mS / cm ~ approx. 30mS / cm, approx. 17mS / cm ~ approx. 3 0mS / cm, approx. 18mS / cm ~ approx. 30mS / cm, approx. 19mS / cm ~ approx. 30mS / cm, approx. 20mS / cm ~ approx. 30mS / cm, approx. 21mS / cm ~ approx. 30mS / cm, approx. 22mS / cm ~ approx. 30mS / cm, approx. 23mS / cm ~ approx. 30mS / cm, approx. 24mS / cm ~ approx. 30mS / cm, approx. 25mS / cm ~ approx. 30mS / cm, approx. 26mS / cm ~ It has an electrical conductivity of approximately 30 mS / cm, or approximately 27 mS / cm to approximately 30 mS / cm. For example, in some embodiments, a solvent for use with mixed-mode chromatography resins is used. The output buffer can have a conductivity of approximately 18 mS / cm to approximately 28 mS / cm.

[0144] In some embodiments, mixed-mode chromatography is used in the method described herein. The elution buffer for use with the resin is an ionic salt, such as NaCl, prepared in approximately 10°C. 0-300 mM (for example, approximately 110-290 mM, 120-280 mM, 130-270 mM) mM, 140~260mM, 150~250mM, 160~240mM, 170~230 It can be included at concentrations of mM, 180-220 mM, or 190-210 mM. For example, the elution buffer for use with mixed-mode chromatography resins is approximately 160 It can have NaCl with a concentration of mM to approximately 240 mM. In a particular embodiment, the present invention Elution for use with mixed-mode chromatography resins in the method described in the details. The buffers are approximately 100mM, 110mM, 120mM, 130mM, 140mM, and 15 0mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280m It contains NaCl at concentrations of M, 290 mM, or 300 mM.

[0145] In some embodiments, mixed-mode chromatography resins are used to create liquid solutions, for example, For example, to purify an anti-α4β7 antibody, such as vedolizumab, from a clarified cell culture recovery product... The method involves adding anti-α4β7 antibody to a column containing a mixed mode resin, at a rate of 40 per liter of resin. Load the column with a protein concentration of ~90, 50~80, or approximately 65g, and the column Wash the column and dissolve it in a solution with a pH of 3.5-4.5, 3.9-4.4, or approximately 4.1. This includes eluting with an elution buffer, such as sodium citrate buffer. In one embodiment, the method is such that the conductivity of the elution buffer is 15-35, 20-30 or This further includes including an ionic salt, such as NaCl, to achieve a density of approximately 24 mS / cm. In some embodiments, the method involves mixing the antibody onto a column containing a resin 1. This includes loading at a protein concentration of 53-77 g per L. Several embodiments The method involves using a solution with a pH of approximately 3.9 to 4.4 and a conductivity of approximately 20 to 28 mS / cm. This includes eluting the antibody from the column using an extraction buffer.

[0146] In some embodiments, the purification of the anti-α4β7 antibody is performed using the mixing mode described herein. Chromatography is used in combination with cation exchange (CEX) chromatography. This can be done by doing so.

[0147] In some embodiments, the methods described herein are mixed-mode chromatography - The yield of anti-α4β7 antibody eluted from the column is determined by one or more para-columns described herein. Compared to the yield of a suitable control process using an elution buffer without a meter, for example If the pH is 3.7 or lower, 3.6 or lower, 3.5 or lower, 3.3 or lower, or 3.0 or lower, then the elution will be... This can be improved compared to the process performed using buffers. In terms of form, the yield is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, the buffer described herein The method involves eluting 50% of the anti-α4β7 antibody from the mixed-mode chromatography column. Above (for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 It can yield recovery rates of 5%, 96%, 97%, 98%, 99%, or higher. In some embodiments, the buffers and methods described herein are used for mixing. 50% to 95% (for example, 5) of the anti-α4β7 antibody eluted from the chromatographic column. 5-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95% This can result in recovery rates of 85-95%, 90-95%, or higher.

[0148] In some embodiments, such compositions using mixed-mode chromatography resins Using the method described above, an anti-α4β7 antibody was mixed with approximately 0% to 2.0% (for example, 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. Compositions can be obtained that contain less than 9% or 2% HMW aggregates. In one embodiment, using such a method with a mixed-mode chromatography resin, anti-α 4β7 antibody and approximately 2% or less (for example, approximately 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, HMW aggregates with concentrations of 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less A composition containing can be obtained. In a particular embodiment, mixed-mode chromatography trees Using a lipid-based method, the levels of anti-α4β7 antibodies were approximately 0% to 2%, less than 2%, and 1.9%. Below, 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, Alternatively, a composition containing 0.1% or less of aggregates can be obtained. In other embodiments, H The level of MW aggregates is at least 1% relative to the level of HMW aggregates in the load material. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% It will be reduced by 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the mixed-mode chromatography method described herein is used. Therefore, the level of HMW aggregates in a composition containing an anti-α4β7 antibody is as described herein. Obtained from a suitable control process using an elution buffer that does not have one or more parameters. Compared to the level of HMW aggregates, for example, pH below 3.7, below 3.6, and below 3.5 The following is a comparison with processes using elution buffers of 3.3 or less, or 3.0 or less. This can be reduced. In some embodiments, the level of HMW aggregates is appropriate. Compared to a control, 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 reduced further.

[0149] Purification of anti-α4β7 antibodies using cation exchange (CEX) resin In one embodiment, the cation exchange (CEX) resin is used in binding / elution mode. Then, a liquid solution containing antibodies and one or more impurities, for example, from clarified cell culture recovery. A method for purifying anti-α4β7 antibodies, such as vedolizumab, is provided. In terms of application, the CEX resin used to purify anti-α4β7 antibodies is a strong cation exchange resin. In certain embodiments, CE suitable for use in the methods and compositions described herein. X resin is -SO3 - It contains functional groups. For example, in some embodiments, the CEX resin is N This is uvia HR-S. The clarified cell culture recovery was recombinantly treated with anti-α4β7 antibody. It can be obtained from host cells that express GS- Chinese hamster ovary (CHO) cells, such as CHO cells or DHFR-CHO cells. It can be converted into cells.

[0150] The CEX method provided herein involves cation exchange chromatography of an anti-α4β7 antibody. This includes bonding to a resin. However, it is not limited to these, including affinity cross-bonding. Mathematics (e.g., protein A chromatography), anion exchange (AEX) Chromatography, mixed-mode chromatography, and hydrophobic interaction chromatography Further purification steps including phi (HIC) are performed before and / or before the CEX method described herein. Or it can be used later. Therefore, in some embodiments, CEX Chroma The load materials used in tography are protein A eluate, AEX eluate, and mixed mo The eluate may include a d-eluate or HIC eluate. Several CEX methods are provided herein. In this embodiment, the CEX resin is washed with a washing solution, and the antibody is eluted from the resin. This can further include: proteins compatible with CEX chromatography, for example. For example, solutions suitable for loading, washing, and eluting anti-α4β7 antibodies are widely available. It is available. In some embodiments, the CEX chromatography solution is citric acid Contains buffers such as salts, acetates, or phosphates.

[0151] In certain embodiments, at least 20 g / L (for example, at least 20 g / L, 25 g) / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 6 0g / L, 65g / L, 70g / L, 75g / L, 80g / L, 85g / L, 90g / L Antibody solutions (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 the CEX resin. In a typical embodiment, the concentration is approximately 25-100 g / L (for example, approximately 25-90 g / L, 25-80 g / L, 25~70g / L, 25~60g / L, 25~50g / L, 25~40g / L, Alternatively, an antibody solution (25-30 g / L) can be loaded into the CEX resin. Specific implementation In terms of form, approximately 25-70 g / L (for example, approximately 25-65 g / L, 30-60 g / L, 35 Antibody solutions of ~55 g / L or 40~50 g / L can be loaded onto CEX resin. For example, an antibody solution of approximately 30-60 g / L can be loaded onto CEX resin.

[0152] The resin should be washed, if necessary, with a suitable washing buffer that does not elute the antibodies bound to the resin. It can be purified. In some embodiments, the washing buffer loads the antibody onto the resin. It has the same composition as the buffer used for that purpose. In one embodiment, the resin is necessary Accordingly, sodium citrate buffer, for example, 25 mM sodium citrate, 50 mM Sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate It can be washed with a water. In some embodiments, the washing buffer has a pH of 5 to 7. pH range, for example, pH 5-6, pH 5.5-6.5, pH 5.1-5.8, pH 5. It has a pH of 3-5.6, pH 6-7, or pH 5.4. It is compatible with CEX chromatography. Other suitable washing buffers are also widely available.

[0153] By adjusting the pH and / or conductivity of the elution buffer, the elution from the CEX resin can be controlled. The level of HMW aggregates in the anti-α4β7 antibody formulation, and the level of the major (primary) isoform species. Adjust the levels of acidic isoform species and / or basic isoform species. It can be restricted. In some embodiments, the CE in the method described herein The elution buffer for use with resin X should have a pH of 6.0 or lower (for example, pH 4.5, pH 4.6, pH4.7, pH4.8, pH4.9, pH5.0, pH5.1, pH5.2, pH5.3, pH5.4, pH5.5, pH5.6, pH5.7, pH5.8, pH5. It has a pH of 9 or less (pH 6.0 or lower). In specific embodiments, as described herein. In this method, the elution buffer used with CEX resin should be approximately pH 4.5 to approximately pH 6 pH of 0 (for example, approximately pH 4.5 to approximately pH 5.8, approximately pH 4.9 to approximately pH 5.9, approximately p H5.0~about pH6.0, about pH5.0~about pH5.9, about pH5.0~about pH5.8, Approximately pH 5.0 to approximately pH 5.7, approximately pH 5.0 to approximately pH 5.6, or approximately pH 5.0 to approximately pH It has a pH of 5.5. For example, the elution buffer for use with CEX resin is approximately It can have a pH of 5.1 to approximately 5.8. In some embodiments, CE The pH of the X elution buffer is the same as that of the washing buffer.

[0154] In addition to or in alternative embodiments described herein, the methods described herein may be used. The elution buffer for use with CEX resin should be approximately 20 mS / cm or less (for example, 19 mS / cm, 18mS / cm, 17mS / cm, 16mS / cm, 15mS / cm, 14 mS / cm, 13mS / cm, 12mS / cm, 11mS / cm, or 10mS / cm It has the following conductivity. For example, the elution buffer for use with CEX resin is 16 It can have an electrical conductivity of mS / cm or less. In some embodiments, as described herein The elution buffer used with the CEX resin in the described method is approximately 10 mS / cm². ~approximately 20 mS / cm (for example, approximately 10 mS / cm to approximately 19 mS / cm, approximately 10 mS / cm) ~Approx. 18mS / cm, approx. 10mS / cm ~ approx. 17mS / cm, approx. 10mS / cm ~ approx. 16 mS / cm, approx. 10mS / cm ~ approx. 15mS / cm, approx. 10mS / cm ~ approx. 14mS / c m, approximately 10 mS / cm to approximately 13 mS / cm, or approximately 10 mS / cm to approximately 12 mS / cm ) has conductivity. In some embodiments, an elution buff for use with CEX resin The material can have an electrical conductivity of approximately 11 mS / cm to approximately 16 mS / cm. Alternatively, or in lieu thereof, the elution buffer for use with CEX resin is 14 mS / It can have an conductivity of less than 1 cm. In certain embodiments, the method described herein In the law, the elution buffer to be used with CEX resin is approximately 11 mS / cm to approximately 14 m S / cm, for example, approximately 12 mS / cm to approximately 14 mS / cm or approximately 13 mS / cm to approximately 14 It has a conductivity of mS / cm. For example, the elution buffer for use with CEX resin is It can have an electrical conductivity of approximately 12 mS / cm to approximately 14 mS / cm. In addition to the above, Alternatively, instead of the above, the elution buffer for use with CEX resin should be 11 mS / cm or higher. (For example, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 m S / cm, 17mS / cm, 18mS / cm, 19mS / cm, or 20mS / cm or less It can have the conductivity shown above. For example, an elution buffer for use with CEX resin. In some embodiments, it can have a conductivity of 12 mS / cm or more.

[0155] In some embodiments, the method described herein is used with CEX resin. The elution buffers are approximately 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM. N at concentrations of mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. It may contain aCl. For example, an elution buffer for use with CEX resin is It can have NaCl concentrations of approximately 90 mM to approximately 120 mM. In a specific embodiment, The elution buffer for use with the CEX resin in the method described herein is approximately 50-150 mM (for example, approximately 50-140 mM, 60-130 mM, 70-120 mM) It has NaCl at a concentration of 80-110 mM or 90-100 mM. Specific implementation In form, an elution buffer for use with CEX resin in the method described herein. - is approximately 70-120 mM (for example, approximately 70-110 mM, 70-100 mM, 70-9 It has a concentration of NaCl (0 mM or 70-80 mM). For example, it is used with CEX resin. The elution buffer for this purpose has a concentration of NaCl of approximately 70 mM to approximately 1100 mM. It is possible.

[0156] In some embodiments, CEX resin is used to create liquid solutions, such as clarified cell cultures. A method for purifying anti-α4β7 antibodies, such as vedolizumab, from nutrient-recovered materials is: The antibody is placed on a column containing CEX resin at a concentration of 40-90, 50-65 per liter of resin, or Load the column with a protein concentration of approximately 57g, wash the column, and then... A buffer with a pH of 5-6, 5.2-5.6, or approximately 5.4, such as sodium acetate buffer. This includes eluting with a fur. In some embodiments, the method involves eluting the elution buffer Ionic salts, e.g., so that the conductivity is 5-25, 10-17, or about 13 mS / cm. For example, further including the inclusion of NaCl. In other embodiments, CEX resin, for example, strong Using cation exchange resins, anti-α4 is extracted from liquid solutions, such as clarified cell culture recovery products. Methods for purifying β7 antibodies, such as vedolizumab, are described in sections 5-6, 5.2-5.6, and It has a pH of approximately 5.4 and an electrical conductivity of 5-25, 10-15, or approximately 13 mS / cm. This includes eluting the column with a buffer, such as sodium acetate buffer. In one embodiment, The method involves using an elution buffer having a pH of approximately 5.4 and a conductivity of approximately 13 mS / cm. This includes eluting ram. In some embodiments, the CEX resin contains approximately 1 liter of resin. Load 57g of protein antibody.

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

[0158] In some embodiments, the CEX method described herein is used to target an anti-α4β7 antibody. and approximately 0% to 2.0% (for example, approximately 0.01%, 0.02%, 0.03%, 0.04%, 0 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0 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 A composition containing % or 2% HMW aggregates can be obtained. Several embodiments In this state, using the CEX resin method, the anti-α4β7 antibody is used at a concentration of approximately 2% or less (for example) For example, approximately 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. Below 0.6%, below 0.05%, below 0.04%, below 0.03%, below 0.02%, and A composition can be obtained that contains HMW aggregates (0.01% or less). Specific embodiments Therefore, using this method with CEX resin, anti-α4β7 antibody was used in concentrations of approximately 0% to 2%, and 2%. Below, 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. Below 0.6%, below 0.05%, below 0.04%, below 0.03%, below 0.02%, and This allows for the creation of compositions containing 0.01% or less of aggregates, such as HMW aggregates. In this embodiment, the level of HMW aggregates is relative to the level of HMW aggregates in the load material. And at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25 %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. The reduction is achieved by using the CEX method provided herein. The level of HMW aggregates in a composition containing an anti-α4β7 antibody is one of the levels described herein. Use an appropriate control CEX process with an elution buffer that does not have the above parameters. Compared to the level of HMW aggregates in the anti-α4β7 antibody formulation obtained by, for example, pH The conductivity is 6.3 or higher, 6.5 or higher, 6.7 or higher, or 6.9 or higher, and / or the conductivity is 18 mS / cm or more, 19mS / cm or more, 20mS / cm or more, 22mS / cm or more, or Compared to processes using elution buffers of 24 mS / cm or higher, the reduction is significant. This is possible. In some embodiments, the level of HMW aggregates is compared with a suitable control. Compared to, 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 so It will be reduced by more than this amount.

[0159] In some embodiments of the methods provided herein, an anti-α4β7 antibody is obtained from a CEX resin. The pH and / or conductivity of the elution buffer used to elute the eluate The isoform distribution of anti-α4β7 antibodies present in the solution can be controlled. For example, The pH and / or conductivity of the output buffer are used to determine the proportion of the primary antibody isoform. Increase the (%) and decrease the percentage of acidic isoform species (%), and / or basic isoform species The percentage of Soform species can be reduced.

[0160] In some embodiments, the value is 6.0 or less, for example, 5.9 or less, 5.8 or less, 5.7 or less. 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, or 5.2 or less, for example, pH Elution bags having a pH of 4.5-6.0, pH 4.5-5.5, or pH 5.0-6.0 A buffer can be selected. In some embodiments, at least 10 mS / cm For example, at least 11 mS / cm, at least 12 mS / cm, at least 13 mS / cm, at least 14mS / cm, at least 15mS / cm, at least 16mS / For example, 10-17 mS / cm, 12-17 mS / cm, 13-17 mS / cm m, 14~17mS / cm, 15~17mS / cm, 16~17mS / cm, 10~16 mS / cm, 12-16 mS / cm, 13-16 mS / cm, 14-16 mS / cm, Alternatively, an elution buffer with a conductivity of 15-16 mS / cm can be selected. In some embodiments, using the above elution buffer conditions, at least 60%, 61% 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71% 72%, 73%, 74%, 75%, or more of the major isoforms of anti-α4β7 antibodies A composition containing foam can be obtained. In some embodiments, the above-mentioned elution buff Using the condition, we set it to 20% or less (for example, approximately 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, Alternatively, a composition containing basic isoform species (or less than 1%) can be obtained. In the application method, the major isoflavone of anti-α4β7 antibody was used with CEX resin. Foam and approximately 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less , 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less , 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less salt A composition containing a basic isoform species can be obtained. In other embodiments, basic isoform species The level of isoform species is less than the level of basic isoform species in the loading material. At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 1 2%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 3 0%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 8 Reduce by 0%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. It will be done.

[0161] III.Analysis method In certain embodiments, chromatography produced using the method described herein. The levels of aggregates, monomers, and fragments in the sample are analyzed. In certain embodiments, aggregates The monomers and fragments were analyzed for each molecule using size exclusion chromatography (SEC). It is measured using, for example, TSK-Gel G3000, although this is not intended to be an exhaustive example. SWxL, 5μm, 125 angstroms, 7.8×300mm column (Tosoh Bioscience) can be used in combination with certain embodiments, and in other embodiments Morphologically, TSK-Gel Super SW3000, 4μm, 250 angstroms, A 4.6 x 300 mm column (Tosoh Bioscience) can be used. In certain embodiments, the above column is Agilent or Shimazhu HP It is used in conjunction with an LC system. In certain embodiments, sample injection is performed, for example, at pH 6. A mobile phase consisting of 100 mM sodium sulfate and 100 mM sodium phosphate was used. The procedure is performed under isocratic elution conditions and detected by UV absorbance at 214 nm. In this embodiment, the mobile phase consists of 1X PBS at pH 7.4, and the elution profile is 28 It is detected by UV absorbance at 0 nm. In certain embodiments, quantification is performed relative to the detected peak. Based on area.

[0162] Use any additional techniques, such as mass spectrometry, to assay size variations. It is possible.

[0163] Various parameters of the antibodies or their antigen-binding moieties reported herein are as follows: It can be measured using standard analytical methods and techniques, such as those described below.

[0164] In different embodiments described herein, cation exchange chromatography (CEX ) Using the antibody or its antigen-binding portion, for example, present in the population of vedolizumab Primary isoforms, basic isoforms (multiple possible), and acidic isoforms (multiple possible). The relative amount of (possible) can be determined. The CEX method determines the antibody species based on the total surface charge. The sample is fractionated. After diluting it to a low ionic strength using a mobile phase, the test sample is placed in an appropriate buffer, e.g. For example, equilibrated with 10 mM sodium phosphate (pH 6.6), for instance, Dionex P ro-Pac(trademark) WCX-10 column (Thermo Fisher Scient It can be injected into CEX columns such as ific, Waltham, MA (USA) The antibody can be eluted using a sodium chloride gradient in the same buffer. Protein elution is monitored at 280 nm, and each peak is identified as acidic, basic, or major isoflavone. It can be assigned to a foam category. The acidic peak is the primary isoform. Elution from the column with a shorter retention time than the peak, and a basic peak, indicates the major isoform. Elution from the column occurs with a longer retention time than the peak. Percentage of major isoforms (%) Report the total percentage of acidic species (%) and the total percentage of basic species (%) of the sample. The suitability of the isoform is determined by comparing its retention time to that of a reference standard. One embodiment The CEX assay method involves diluting the test sample to a low ionic strength and using 10 mM ri Inject into a CEX column equilibrated with sodium phosphate (pH 6.6), and this buff Elute the column with an NaCl gradient in the ferrite and monitor the peak at 280 nm. This includes assigning each peak as acidic, primary, or basic, and the acidic peak The first peak eluted with the shortest retention time, the second peak eluted, and the basic peak eluted with the longest retention time. Elution occurs over time, the area of ​​each peak is quantified, and the amount is expressed as a percentage of the total peak area. It is calculated as follows.

[0165] In the different embodiments described herein, size exclusion chromatography (CEX) Using this, the antibody or its antigen-binding portion, for example, the mo in the vedolizumab population, is identified. Determine the relative levels of nomers, high molecular weight (HMW) aggregates, and low molecular weight (LMW) degradation products. It can be determined. The SEC method is used to determine antibody monomers from HMW species and LMW degradation products. This enables separation based on ISIS. Each test sample and reference standard is separated using an appropriate buffer. The analysis can be performed using commercially available SEC columns. For example, in some embodiments... SEC analysis is performed using a G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA), or two G3s connected in series 000 SWxl column and isocratic sodium phosphate-chloride buffer This can be done using a stem (pH 6.8). Protein elution can be monitored at 280 nm. Observe the sample. The purity is determined by evaluating the main peak (monomer) and the total peak area. One embodiment For SEC analysis, the sample is injected into two G3000 SWxl columns connected in series. This involves using an isocratic sodium phosphate-sodium chloride system (pH 6.8) The process involves flushing, and the elution of protein species is monitored at 280 nm, with the main peak being (monomer). ) and the total peak area are measured. Sample purity (%) (calculated as monomer (%)), H Report the percentage of MW aggregates and / or LMW degradation products.

[0166] Residual CHO host cell protein (HCP) impurities present in antibody preparations are removed as needed. Therefore, it is measured by plain-bonded immunosorbent assay (ELISA) using standard techniques. This is possible. Cygnus Technologies (Southport, NC(U CHO HCP ELISA kit 3G sold by SA)) is designed for this purpose. Many ELISA kits are commercially available. Host cell proteins in the test sample are solid. It can then be captured using a standardized polyclonal anti-CHO HCP antibody. The captured protein is then subjected to the same antibody labeled with horseradish peroxidase, etc. It can be detected using a suitable detection reagent. In this exemplary embodiment, CHO H The amount of captured peroxidase, which is directly proportional to the concentration of CP, is determined by the peroxidase substrate. Using 3,3',5,5'-tetramethylbenzidine (TMB), colorimetric analysis was performed at 450 nm. It can be measured by analysis. Therefore, the CHO HCP assay is polyclonal This involves capturing HCP using an anti-CHO HCP antibody, where HCP is peroxy The 3,3',5,5'-tetramethylbenzidine (TMB), which is the dase substrate, is at 450 nm Polyclonal anti-CHO HCP antibody is converted into a substrate that can be measured by colorimetric analysis. It is detected after binding with an antibody labeled with horseradish peroxidase. The concentration of HCP is, This can be determined by comparing it with the CHO HCP calibration curve, which includes the components of the test kit. It is reported as the percentage (%) of the total protein level in the antibody preparation.

[0167] IV. Downstream Processes and Formulation Anti-α4β7 antibody (e.g., vedolizumab or having a binding domain equivalent to vedolizumab) The antibody can be further purified from the contaminating soluble proteins and polypeptides. Examples of suitable purification methods include the following, which can be used individually or in combination as needed. Furthermore, it can be used in conjunction with one or more of the methods provided herein: for example, Affinity chromatography using resins that bind to the Fc region of antibodies such as protein A. Raffy; for example, SP-Cephalose(trademark) or CM-Cephalose(trademark) Hidden Ion exchange chromatography (CEX) such as cation exchange chromatography of roxyapatite Fractionation using ram or resin; anion exchange chromatography (AEX); hydrophobic interaction Action chromatography (HIC); mixed-mode chromatography; ethanol precipitation; Chromatography focusing; ammonium sulfate precipitation; e.g., Sephadex G-75 ( Gel filtration using (trademark); ultrafiltration and / or diafiltration, or the above A combination of these. An example of a purification method is Liu et al., mAbs, 2:480-4 It is described in 99 (2010). At the end of the purification process, the purity of the recombinant protein The degree of purity is high, and for example, it is suitable for human therapeutic applications in the pharmaceutical antibody preparations described below. After preparation, the highly purified recombinant protein is subjected to ultrafiltration / diafiltration (UF / DF). ) can be performed to create a pharmaceutical formulation suitable for administration to humans.

[0168] After ultrafiltration / diafiltration, the antibody preparation may remain in liquid form, It may be freeze-dried to obtain a dried antibody preparation. In one embodiment, the dried freeze-dried antibody preparation is 180mg, 240mg, 300mg, 360mg, 450mg, or 600mg of anti-alpha It is supplied in a single-dose vial containing 4β7 antibody and is administered after reconstitution with a liquid such as sterile water. This can be done. In another embodiment, an anti-α4β7 antibody, such as vedolizumab, is administered to the target population. Until administered, it should be kept in a container such as a vial, syringe, or cartridge for approximately 2-3 days. It is contained in a stable liquid pharmaceutical composition that is stored at 8°C. In some embodiments, it is an anti-α4β 7. The lyophilized formulation or stable liquid pharmaceutical composition of the antibody contains approximately 0% to 5.0%, 0 Contains aggregates of %~2%, 2% or less, 1% or less, 0.6% or less, or 0.5% or less.

[0169] Therefore, in some embodiments herein, a humanized anti-α4β7 antibody, or A lyophilized or stable liquid pharmaceutical formulation of the anti-α4β7 antibody containing the antigen-binding portion. A composition is provided. An example of a lyophilized formulation containing an anti-α4β7 antibody, such as vedolizumab, is: This information is provided herein by reference to U.S. Patent No. 9,764,033, which contains the same information. For reference, an example of a liquid formulation containing an anti-α4β7 antibody, such as vedolizumab, is listed in the U.S. Patent No. 10. It is described in publications 040,855, and its contents are incorporated herein by reference. In several embodiments, the lyophilized or stable liquid formulation of the anti-α4β7 antibody is used. The composition is approximately 11%-16%, 12%-15%, 14% or less, 13% or less, and 12% or less. or containing 11% or less of basic isoform species. In some embodiments, anti-α4β 7. The lyophilized antibody formulation or stable liquid pharmaceutical composition contains 65% to 75%, 66%. %~74%, 67%~73%, at least 65%, at least 66%, at least 67% , at least 68%, at least 69%, or at least 70% of the primary isoforms Includes.

[0170] Purified antibodies, for example, anti-α4β7 antibodies (e.g., vedolizumab or vedolizumab An antibody having a binding region corresponding to the above is concentrated to obtain a concentrated protein composition, for example, At least 100 mg / mL, 125 mg / mL, or 150 mg / mL of antibody Concentration, or approximately 100 mg / mL, 125 mg / mL, or 150 mg / mL A protein composition having a concentration can be provided. The concentrated antibody product is concentrated in a concentration field. Concentrate to a level possible under these circumstances, for example, to a concentration where the polypeptide is no longer soluble in the solution. It should be understood that this is possible.

[0171] In some embodiments, the compositions obtained herein, such as purified vedolizumab, are used. It contains an anti-α4β7 antibody, which is then formulated for use in humans. In one embodiment, The purified antibodies are then formulated as a dried, lyophilized preparation, and then mixed with a liquid such as sterile water. It can be returned and administered. The returned preparation can be administered via one of the routes described above. It can be administered by parenteral injection. Intravenous injection can be administered by sterile isotonic saline, bac For example, phosphate-buffered saline or Ringer's solution (lactate or dextrose). By further dilution with a liquid, it can be administered by intravenous drip. Several embodiments Then, the purified antibody is formulated as a liquid formulation, and the anti-α4β7 antibody is, for example, approximately 54m It is administered by subcutaneous injection in doses of g, 108 mg, 165 mg, or approximately 216 mg. .

[0172] The purified compositions described herein can be used for storage and freezing. The containers used are polycarbonate bottles (for IV preparations) or PETG bottles (for subcutaneous preparations). Examples include (for use as a pharmaceutical agent). After dispensing the formulation into bottles, it can be frozen (for example, - Below 60℃).

[0173] The following examples illustrate improved methods and compositions for purifying antibodies. The purified composition of anti-α4β7 antibodies, particularly vedolizumab, was obtained using the following Examples 1-5. You can obtain the object. This specification describes various parameters as described in the following examples. This includes the methods described in the following examples, which include the following:

[0174] Examples In the following examples, vedo produced in cell cultures using CHO cells as an expression system This section describes the purification process for lizumab.

[0175] Example 1: Elution buffer for vedolizumab purification using protein A resin influence In this embodiment, it is used to manufacture a therapeutic anti-α4β7 antibody, for example, vedolizumab. This document describes an antibody purification method using protein A resin, which can be performed as described herein. By adjusting the elution pH of the protein A resin, aggregates in the purified vedolizumab composition can be removed. The level has decreased.

[0176] Vedolizumab was used in recombinant Chinese hamsters genetically engineered to express antibodies. - Manufactured by cell culture of ovarian (CHO) cells (GS-CHO) (general cell culture method) For more information, see Li et al. (2010) mAbs 2:5, 466-477. ).

[0177] After cell culture in CHO cells, primary recovery was performed using a protein A affinity column. Subsequently, selective capture of vedolizumab was performed. Affinity using recombinant protein A resin. From the clarified recovered material obtained from the upstream primary recovery process by performing chromatography Antibodies were selectively removed. In this step, the process of host cell proteins (HCPs) and other processes was performed. Impurities related to sap were also removed.

[0178] The protein A resin was first equilibrated in PBS equilibration solution (pH 7.2). Then, The clarified recovered material was loaded. Three washes were performed. Wash 1 was a PBS wash using the same equilibration solution. The wash was performed in a solution (pH 7.2), and the second wash was performed in a 1M NaCl PBS washing solution (pH 7.2). Wash 3 was performed using the same solution as used for Wash 1 (PBS) and equilibration. The antibody was derivated. Because they remained bound to the lipids, each wash served to remove impurities from the antibodies. Next Then, antibodies were eluted from the resin using elution buffers with different pH levels. This is shown in Figure 1. We tested elution buffers with pH 3-3.5 for sea urchin. The results shown in Figure 1 indicate that higher pH values ​​were effective. This indicates that the percentage of aggregates decreases as p When vedolizumab is eluted from protein A using H3, high levels (i.e., approximately 1-1.2%) are observed. An eluate with aggregates at a higher pH (for example, pH approximately 3.) was obtained. The eluate obtained by eluting the antibody using an elution buffer containing 5) is approximately 0.6-0. It contained 85% aggregates.

[0179] Further testing will be conducted to address the purification of protein A, which significantly impacts the product quality of vedolizumab. Related process parameters were identified. GS-C expresses vedolizumab by recombination. The clarified and recovered product from HO cells is MabSelect SuReLX resin (GE Heal The bound antibody was loaded into (thcare, Pittsburgh, PA). Washed standing with sodium citrate buffer and PBS. Elute pH was set to pH 3.3~p Evaluation was performed within the range of H3.9. Sodium citrate buffer was used for elution. Aggregates The level (percentage of HMW species (%)) and the level of HCP in the purified vedolizumab composition The effects of the elution buffer are shown in Table 1 and Figure 2.

[0180] As shown in Table 1, the linear regression model showed that elution pH was significant for all assay results. This showed that it has a significant effect (p<0.05). The variability of the data is due to the LMW ratio (%) and The HCP was slightly larger, but the load amount affected the HCP clearance. The combination of volume and load flow had a slight effect on LMW (%).

[0181] ·monomer(%): The elution pH had a significant effect on the monomer percentage (%) (p<0.05). The input parameters also did not show a correlation with the monomer percentage (%). Consequently, the monomer percentage increased.

[0182] • HMW share (%): The elution pH had a significant effect on the HMW percentage (%) (p<0.05). The force parameters also did not show a correlation with the HMW ratio (%). As a result, the percentage of HMW vehicles decreased.

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

[0184] HCP: Elution pH and load have a significant effect on HCP in terms of both ppm and logarithmic reduction coefficient. This was shown (p<0.05).

[0185] [Table 1] Vedolizumab is superior to other IgG antibodies, for example. It possesses properties such as high hydrophobicity. Figure 3 shows a comparison of vedolizumab with three other IgG antibodies. This shows each antibody (vedolizumab (MLN0002), IgG A, IgG B, or I Using a pH-increasing elution buffer (pH increasing from left to right), gGC) was used. Elution obtained when eluted from a cation exchange column (Nuvia S, Bio Rad) This shows the amount of aggregates found within (HMW percentage (%)). Vedolizumab aggregate clearance. The performance variation of the sample was greater than that of the other three tested IgG samples under their respective optimal conditions. It was large. Therefore, as can be seen in Figure 3, pH was important for aggregation during the purification of vedolizumab. It can have an effect on the body level.

[0186] Example 2: Purification of vedolizumab using hydrophobic HIC resin Considering the hydrophobic properties of vedolizumab, reducing HMW aggregates is a downstream process. In some cases, this can be difficult with Seth. Furthermore, vedolizumab can be used in mammalian cells such as CHO cells. When produced within a cell, it can also minimize the level of host cell proteins (HCPs). It is essential. HIC, mixed mode, and anion exchange resin and membrane, high throughput The performance was screened using the following method. After this, eight types of HIC resins were tested on a vedoor. Regarding the ability to reduce aggregates and minimize HPC in the purification of zumab, Tests were conducted under different equilibration, loading, and elution conditions. The acceptable aggregate clearance was shown. And the only resin that was able to minimize HCP was Toyopearl Hexyl- 650C (Tosoh Biosciences) was a hydrophobic HIC resin. More specifically... In particular, Hexyl-650C is suitable for binding under appropriate conditions (for example, 0.5M at pH 6.7). (NH4)2SO4) reduces the aggregate level to approximately 1.5% to 0.35% of HMW aggregates. Other resins tested included butyl-650M, butyl-600M, and superbutyl. -55C, phenyl-650M, phenyl-600M, PPG-600M, and ether Although -650M was included, such low levels of aggregates could not be achieved.

[0187] Hexyl-650C contains ether, PPG, phenyl, and butyl, and has been tested. It was the most hydrophobic resin compared to other resins. Hexyl-650C is approximately 1000A. It has an average pore size and an average particle size of approximately 100 μm.

[0188] Further experiments will be conducted using Hexyl-650C in binding / elution and flow-through modes. Both methods were used. In the binding / elution experiment, Hexyl-650C formed aggregates with an HMW of approximately 0.3 It was possible to reduce it to 0%, but the binding capacity was low (approximately 20 mg per 1 ml of resin). In contrast, a flow-through mode using Hexyl-650C and vedolizumab... Then, both a decrease in aggregates and an increase in load capacity were observed. 10 mM sodium phosphate Initial unadjusted load of 108 mg / ml in 0.2 M sodium chloride in a solution of 6.7 (pH 6.7) A flow-through experiment was conducted using the following conditions. Under these flow-through conditions, HMW was 1.39%. This resulted in a reduction to ~HMW 0.71%. The reduction of aggregates was further improved by increasing the salt content, including the conversion factor. 250 mM phosphorus 67.5 mg / using potassium acid and 50 mM potassium chloride (for equilibration and load adjustment) A smaller load of ml reduces aggregates from approximately 0.72% to approximately 0.3% of the HMW. This resulted in a recovery rate of 95.5%.

[0189] A column-based design of experiment (DOE) test was performed to determine the degree of condensation during the purification of vedolizumab. The ability of the Hexyl-650C in flow-through mode to reduce aggregate levels Further evaluation was conducted. As shown in Table 2, low pH and high phosphate load equilibration conditions were used. In this case, a low HMW ratio (%) and low HCP levels (ppm) were obtained. 60m The experiments shown in Table 2 were performed using a resin load of g / ml. Under low pH conditions, all HMWs The reduction rate was between 1.0% and 0.34% or less of HMW. The average antibody recovery rate was approximately 91%. It was 0.5%. The combination of high pH and high potassium phosphate is a major species of vedolizumab. It appears that the affinity for the resin increased, and the recovery rate decreased. In contrast, a higher The combination of low phosphate levels and low pH resulted in increased HMW clearance. Phosphates and low pH result in low HMW, low HCP, and low residual protein A exudation. Observed (see, for example, line 12 below). Therefore, the highly hydrophobic HIC resin does not coagulate. Effective removal of cluster (HMW) vedolizumab to levels lower than 0.5% Done.

[0190] [Table 2]

[0191] Example 3: Purification of vedolizumab using mixed-mode chromatography resin Effects of column loading, elution buffer pH, and conductivity We created a production CHO cell line that expresses vedolizumab at a high antibody titer (≧5.0 g / L). This production CHO cell line was designed to cope with large amounts of this highly hydrophobic antibody. The development of a purification process is needed.

[0192] Capto Adhere ImpRes provides strong anionic bonding with smaller bead diameters. Mixed-mode (MXM) chromatography with functional exchange, hydrogen bonding, and hydrophobic interactions. It is a resin that allows for improved impurity removal and increased capacity.

[0193] Capto Adhere ImpRes mixed mode used in flow-through mode The resin was capable of purifying vedolizumab, but the yield and level of impurity removal were It was lower than ideal. Capto Adhere ImpRes was used in binding-elution mode. Prior characterization experiments conducted using the following methods revealed the resin load capacity, the pH of the elution buffer, and Step yield and / or related to the three process input parameters of conductivity of the elution buffer Alternatively, a significant loss in impurity removal performance was observed. In this example, changing these parameters... The dynamics affect the performance of Capto Adhere ImpRes in vedolizumab purification. Further investigation will be conducted into the effects of this, as well as its impact on various product quality characteristics. Let's discuss the planned tests.

[0194] Materials and methods The clarified cell culture solution was purified with protein A, and then applied to CaptoAdhere. ImpRes (GE Healthcare, Chicago, IL, USA) Chromatography The resin was loaded onto the Graphy column. The mixed-mode resin was mixed with sodium phosphate in a pH 7.8 solution. The columns were washed using a fur filter, and antibodies were eluted from the column under various conditions described below.

[0195] The samples were immediately submitted for analysis by SEC, stored at 2-8°C, and processed within one week. The remaining assays (CEX, CHO HCP ELISA) are stored frozen (-80°C). The analysis was performed using (°C). The method used for the analysis is shown in Table 3 below and described in detail below. The quality of the load material was determined by analyzing samples taken after the load material was placed in the queue and then placed into the queue. We confirmed that there were no significant changes in the characteristics.

[0196] [Table 3]

[0197] Experimental Design A complete factorial design is implemented using three factors: resin load amount, elution buffer pH, and elution buffer conductivity. It was used at two levels. Sodium citrate elution buffer was used in these experiments. The proposed plan included 30 trials with three central point conditions. Further central point conditions This was included as part of the experimental design shown as DOE pattern 222 in Table 5. Process input parameters were maintained at the center point conditions. Sodium chloride concentration of elution buffer. The experiment was planned using the degree, and the conductivity measurement value of the elution buffer was used as an input parameter value for statistical analysis. It was used as follows. The tested parameter ranges and the outline of the plan are described in Tables 4 and 5, respectively. do.

[0198] [Table 4] [Table 5] DOE pattern: (3): Upper level, (2): Middle level, (1): Lower level, (0) : The center point of each input parameter range. The experiment was conducted using NaCl concentration as an input parameter. The actual conductivity measurements were then used in the statistical analysis.

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

[0200]

number

[0201] statistical analysis Product quality, showing assay results and KPIs for all experiments in the plan, is defined in JMP 1. 1. Analyze using statistical software (SAS Institute, Cary, NC) Each response was analyzed by fitting it to the linear model shown in Equation 1.

[0202] Equation 1 General linear model of regression

number

[0203] In statistical analysis and modeling, a relatively small dataset with a relatively large number of potential inputs Applying this to a model often results in overfitting. One of the features of the fitting is high R 2 It has values ​​but multiple scientifically meaningless (and statistically) This model has terms that are not statistically significant. To determine a statistically significant model, forward regression of the input parameters and the desired response are used. We developed each model. The regression stopping rule is a p-value threshold, and input is stopped if the p-value < 0.05. The parameters were incorporated into the model. This analysis algorithm determines (i) the highest Possible R 2 (ii) to achieve the value, and include as few input parameters as possible, (iii) This describes the physically possible behavior of antibodies subjected to multimode chromatography. (Even if such findings do not appear to match the initial technical expectations), the model was generated. Generated.

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

[0205] [Table 6]

[0206] [Table 7]

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

[0208] FIGS. 4 and 5 show plots of the process recovery rate by Capto Adhere ImpRes against the pH and conductivity of the elution buffer or the loading amount. The recovery rate was significantly affected by the pH of the elution buffer (p < 0.0001) and the resin loading amount (p = 0.0021), and although not as much, also by the conductivity of the elution buffer (p = 0.0189). At an elution buffer pH of about 4.40 for any level of elution buffer conductivity and loading amount, the process recovery rate was 83.91% or less (Runs 1, 4, 11~ 0.0189). At an elution buffer pH of about 4.40 for any level of elution buffer conductivity and loading amount, the process recovery rate was 83.91% or less (Runs 1, 4, 11~ 14, 16, 19, and 27). Low resin loads negatively impacted recovery rates. In the washing process after sample loading, all of the load amount of 77g per liter of resin was processed. A breakthrough was observed in the row. The effect of the load amount was dependent on the elution buffer pH. p=0.0170). At low elution buffer pH (i.e., pH 3.8), load While the quantity did not have a substantial impact on the recovery rate, the elution buffer pH increased. Consequently, the recovery rate decreased with a low resin load (at an elution buffer pH of approximately 4.4). For a load of 77g per liter of resin, the percentage is 81.94-83.91%, compared to per liter of resin With a load of 53g, the conductivity was 73.36-78.84%. The conductivity of the elution buffer was experimental. The results and model predictions indicated that it had only a slight impact on the recovery rate. Elution buffer pH 4.40, elution buffer conductivity 28.89 mS / cm, and load per 1L of resin. With a quantity of 53g, the lowest recovery rate was observed at 73.36% (Trial 13, model 76%). (Predicted to be 0.22%).

[0209] Therefore, as shown in Figures 4 and 5, the yield of vedolizumab is mixed mode chloro The matrixing resin is used with an elution buffer having optimized pH and conductivity. The rate increases when this occurs.

[0210] Effect of pH and conductivity of elution buffer on HMW aggregates The effect of pH and conductivity of the elution buffer on the level of aggregates is shown in relation to the input HMW amount. The effects of force parameters are shown in Figures 6 and 7.

[0211] According to the linear regression model, the amount of HMW species in the eluate is determined by the pH of the elution buffer (p<0 (0.0001), conductivity of the elution buffer (p<0.0001), and load amount (p=0. (0015) is affected by (R 2 =0.962). When the elution pH and conductivity increase, The amount of HMW species in the eluate decreases, and the level increases as the load increases. The elution buffer showed a statistically significant (p=0.0462) relationship between pH and conductivity. ) Interactions were also predicted.

[0212] As shown in Figures 6 and 7 below, the HMW species content of approximately 1% or more is approximately 3.80. Observed at efflux pH. Elution pH 3.80, elution conductivity 19.67 mS / cm(16 With 0 mM NaCl and a load of 65 g per 1 L of resin (experiment 2), the maximum concentration was 1.23%. A high HMW content was obtained (predicted to be 1.19% by the model). In the worst-case scenario, the HMW content was 1.24% at 77 g / L under the same elution buffer conditions. .

[0213] According to the linear regression model, the elution buffer pH (p<0.0001), elution buffer Conductivity (p=0.0003) and load amount (p=0.0016) are respectively determined by HMW. It has a statistically significant impact on clearance performance. It remained constant under all conditions evaluated in this test. A clearance level of (12.50-72.79%) was achieved among these inputs. However, the elution pH had the biggest impact. When the elution buffer pH increased, HMW crystals... Although the elution rate improved, this had the opposite effect on the recovery rate. According to the model, elution Increasing the buffer conductivity and decreasing the load resulted in increased HMW clearance.

[0214] Therefore, as shown in Figures 6 and 7, aggregates in the purified vedolizumab composition The level (percentage of HMW species (%)) is determined by the elution of antibodies from the mixed-mode chromatography resin. Adjusted by selecting the pH and conductivity of the elution buffer used. It is possible. Furthermore, the level of aggregates is such that mixed mode chromatography resins Decreases when used with elution buffers having a high pH and / or high conductivity. It is possible.

[0215] Example 4: Elution batch for vedolizumab purification using cation exchange (CEX) The influence of fur As a means of further reducing the level of aggregates in vedolizumab formulations, cation exchange ( CEX chromatography was also investigated. This specification describes Nuvia HR-S tree CEX chromatography using lipids (Bio-Rad, Hercules, CA, USA) Regarding the study focused on adapting this hydrophobic antibody to the purification of vedolizumab, This report focuses particularly on reducing aggregate levels from the sample. CE was performed in binding / elution mode. The elution conditions for process X were evaluated. Using a design of experiments (DoE) approach, the elution batch was evaluated. Several parameters, including the pH of the elution buffer and the conductivity of the elution buffer, are related to the outcome of the process. The impact on [the subject] was evaluated.

[0216] Materials and methods The load material and analytical method used in this test are the same as those described in Example 3 above.

[0217] Experimental Design Initial screening tests and preliminary risk assessments indicated that the resin would not be used in binding / elution mode. When used, the pH and conductivity of the elution buffer are as follows: Nuvia HR - Known or potential impacts on the process performance results (PPO) of -S This was identified. This study was conducted to characterize the effects of these process parameters. The parameters and experimental design for the test are shown in Tables 8 and 9, respectively. The conductivity of the buffer can be changed by adjusting the concentration of sodium chloride (NaCl). The range of NaCl levels evaluated is shown in Table 8.

[0218] [Table 8]

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

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

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

[0222] As shown in Figures 8 to 10, the variations in HMW, monomer, and LMW of the eluate are as follows: These are 0.01-0.88%, 98.33-99.27%, and 0.62-1.35%, respectively. Yes, HMW's model has a strong linear dependence on the pH and conductivity of the elution buffer. , along with the interaction term which includes both parameters. The model surface (shown in Figure 8) is HMW is lowest at the pH and conductivity values ​​of the elution buffer with extremely low HMW. This indicates the highest values ​​for extremely high elution buffer pH and conductivity.

[0223] To explain the variation in HMW content of the load material throughout the entire test, HM was provided for each application. W clearance was measured. Similar to the HMW of the eluate, the HMW clearance was measured throughout the test. The HMW clearance model fluctuates significantly (-30.65 to 98.61%), and the elution rate is high. The pH and conductivity of the buffer include linear and interaction terms. Figure 9 shows the model. This behavior is observed. The highest HMW clearance is observed under conditions of low pH and conductivity of the elution buffer. While a value of 'S' was obtained, HMW clearances exceeding 70% were observed under many test conditions. This was shown. However, the results for trials 9, 20, and 24 (-1.18% and -20%, respectively). Negative HMW clearance values ​​reported for 55% and -30.65% are aggregates Large variability in the removal rate was observed across a wide range of conditions evaluated, and under specific conditions... This indicates that aggregate species may not be removed, but rather generated.

[0224] In trials 40-42, the load material was generally used in the CEX process under center point conditions. Capto Adhere ImpR containing a higher aggregate level than those typically used. The es eluate was used as the load material. The HMW and HMW clearance models were soluble By increasing the pH and conductivity of the elution buffer, an eluate with a high HMW content can be produced. This demonstrated that the conditions selected for trials 40-42 were the "worst" conditions for CEX load materials. The aim is to explore potential elution conditions using aggregate levels in the case of " The pH value of the elution buffer is 5.50 to 5.54 and the elution buffer is 13.40 mS / cm. Based on the conductivity of Farr, the obtained eluate HMW was 0.31-0.34%. However, However, the eluate HMW increased to 0.59% when using an eluate with a pH of 5.60 (compared to (And the conductivity did not change). At the aggregate level, further processes are performed in HMW. Therefore, there was a possibility that the patient did not meet the acceptable criteria for vedolizumab.

[0225] LMW is linear with increasing pH or conductivity of the elution buffer. It was shown to decrease. This model includes the elution buffer pH / elution buffer conductivity. The relationship between elution buffer pH / load volume and elution buffer conductivity / load volume The section on action was also included. Similar to the model for HMW, the monomer model is elution buffer The strong dependence of the pH and conductivity was included in the form of linear and interaction terms. The Dell surface (shown as a saddle function in Figure 10) has an extremely high pH of the elution buffer and This indicates that the monomer with the lowest conductivity is obtained when the conductivity conditions are combined.

[0226] Therefore, as shown in Figures 8 to 10, the pH and conductivity of the elution buffer are used The level of aggregates (H) in the composition containing vedolizumab purified using CEX resin. The percentage of MW species can be adjusted. Furthermore, as shown in Figures 8 to 10... The level of aggregates in the purified vedolizumab composition is low in the CEX resin and / or This can be reduced when used with an elution buffer that has low conductivity.

[0227] Effect of pH and conductivity of elution buffer on basic isoform species The effects of pH and conductivity of the elution buffer on the level of basic isoform species are investigated by acid Figure 1 shows the effect of input parameters on the content of sex, major, and basic isoform species. Figures 1 through 13 are shown.

[0228] As shown in Figures 11-13, the results for acidic, primary, and basic content are as follows: 12.49-30.27%, 64.32-73.82%, and 5.42-18.04% It was within the range. The model for acid content is based on the pH of the elution buffer and the elution buffer's It includes conductivity and load, along with terms representing the interaction of all three parameters. The pH and conductivity of the elution buffer had the strongest influence on the acidity. As seen on the model surface in Figure 11, the highest acidic content corresponds to the pH of the elution buffer and the elution buffer. This was obtained when using a combination of values ​​with extremely low power efficiency.

[0229] The model developed for the content of the major isoforms involves the pH of the elution buffer and the elution buffer. This includes terms for the interaction between the conductivity of the buffer and the load amount, and the pH / solution of the elution buffer. The interaction of the conductivity of the output buffer has the greatest influence on the model behavior. (See Figure 12) As shown, the highest major isoform content corresponds to the highest elution buffer conductivity and Obtained with the lowest elution buffer pH, and with the lowest major isoform content. However, this is predicted by the extremely low pH and conductivity combination of the elution buffer.

[0230] The pH of the elution buffer, the conductivity of the elution buffer, and a linear term of the load amount are related to the eluate. The basic isoform content is most strongly influenced, while the contribution of the interaction term is small. As seen on the model surface in Figure 13, the basic isoform content is in the elution buffer. It increased in response to increases in pH and conductivity of the elution buffer.

[0231] Therefore, as shown in Figures 11 to 13, the pH and conductivity of the elution buffer are used. Furthermore, charged isoflavones in a composition containing vedolizumab purified using CEX resin The foam distribution can be adjusted. As shown in Figure 11, the purified composition of vedolizumab. The level of acidic isoform species in the material is high pH and / or high conductivity of CEX resin. Furthermore, this can be reduced when used with an elution buffer containing [the specified component]. As shown, the levels of basic isoform species in the purified vedolizumab composition are C When EX resin is used with an elution buffer having a low pH and / or low conductivity It can be reduced to this extent.

[0232] Example 5: Determination of product quality characteristics The following analytical assays and methods are used to determine the product quality characteristics of vedolizumab. It was used in the case study.

[0233] Cation exchange chromatography (CEX) is a method of vedolism based on the overall surface charge. The antibody species (major isoforms, basic species, and acidic species) are fractionated. Low-temperature analysis is performed using a mobile phase. After dilution to ionic strength, the test sample was equilibrated in 10 mM sodium phosphate (pH 6.6). Modified Dionex Pro-Pac(trademark) WCX-10 column (Thermo Fi Sher Scientific, Waltham, MA (USA)) injected the same Protein elution was performed using a sodium chloride gradient in a buffer. Protein elution was monitored at 280 nm. We then analyzed the data and assigned each peak to the category of acidic, basic, or major isoform. The percentage of major isoforms (%), the total percentage of acidic species (%), and the percentage of basic species (%). Report the total of the following: Compare the retention times of the primary isoforms of the sample to the reference retention times. The suitability is then determined.

[0234] The purity of vedolizumab is determined using size exclusion chromatography (SEC). The reference and test sample (75 μg) were connected in series to two G3000 SWxl columns. Tosoh Bioscience,King of Prussia,PA (USA) ) and an isocratic sodium phosphate-sodium chloride buffer system (pH 6.8) The analysis is performed using [method name]. This method analyzes high molecular weight (HMW) species and low molecular weight (LMW) degradation products. This provides separation of antibody monomers from [the source]. Elution of protein species is monitored at 280 nm. Observe the sample. Determine the purity by evaluating the main peak (monomer) and the total peak area. Sample purity Report the percentage of monomers (%) and aggregates (%).

[0235] Equal portions Those skilled in the art will recognize or confirm many equivalents of the specific embodiments of the inventions described herein without performing any experiments beyond the usual procedures. Such equivalents are included in the following claims. All references, patents, and published patent applications cited throughout this application are incorporated herein by reference. The present invention includes the following embodiments. [1] A method for obtaining a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The anti-α4β7 antibody is bound to the protein A by contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a matrix containing protein A. Washing the matrix containing protein A with a washing solution, The process involves contacting the matrix with an elution solution having a pH of 3.2 to 4 to elute the anti-α4β7 antibody from the matrix containing protein A, thereby obtaining a composition containing the anti-α4β7 antibody. The method wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region including the CDR3 domain described in SEQ ID NO: 4, the CDR2 domain described in SEQ ID NO: 3, and the CDR1 domain described in SEQ ID NO: 2, and a light chain variable region including the CDR3 domain described in SEQ ID NO: 8, the CDR2 domain described in SEQ ID NO: 7, and the CDR1 domain described in SEQ ID NO: 6. [2] The method according to [1] above, wherein the composition containing the anti-α4β7 antibody contains less than 1% high molecular weight (HMW) aggregates. [3] The method according to [1] or [2] above, wherein the protein A is immobilized on a solid phase. [4] The method according to [3] above, wherein the solid phase comprises one or more of beads, gels, and resins. [5] The method according to any one of the above [1] to [4], wherein the pH of the washing solution is approximately 7. [6] The method according to any one of the above [1] to [5], wherein the elution solution contains citric acid. [7] The method according to any of the above [1] to [6], wherein the pH of the elution solution is 3.2 to 3.7 or 3.3 to 3.8. [8] A method for obtaining a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The method involves obtaining a composition containing the anti-α4β7 antibody by contacting a solution containing the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-α4β7 antibody to flow through the HIC resin. The aforementioned HIC resin is characterized as a highly hydrophobic HIC resin. The method wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region including the CDR3 domain described in SEQ ID NO: 4, the CDR2 domain described in SEQ ID NO: 3, and the CDR1 domain described in SEQ ID NO: 2, and a light chain variable region including the CDR3 domain described in SEQ ID NO: 8, the CDR2 domain described in SEQ ID NO: 7, and the CDR1 domain described in SEQ ID NO: 6. [9] The method according to [8] above, wherein the composition comprises the anti-α4β7 antibody and contains less than 0.6% HMW aggregates.

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

[11] The method according to

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

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

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

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

[12] , wherein the composition contains less than about 0.22 ppm of residual protein A.

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

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

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

[14] , wherein the highly hydrophobic HIC resin has an average pore size of about 50 to 150 μm.

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

[14] , wherein the highly hydrophobic HIC resin has an average pore size of about 100 nm and / or a pore size of about 100 μm.

[17] A method for producing a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The anti-α4β7 antibody is bound to the resin by contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin. Washing the mixed mode chromatography resin with a washing solution, The method includes obtaining a composition containing the anti-α4β7 antibody by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby eluting the anti-α4β7 antibody from the mixed-mode chromatography resin. The method comprising the anti-α4β7 antibody, the heavy chain variable region sequence described in SEQ ID NO: 1 and the light chain variable region sequence described in SEQ ID NO: 5.

[18] The method according to

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

[19] The method according to

[17] or

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

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

[17] to

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

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

[17] to

[20] , wherein the eluting solution has an electrical conductivity of 30 mS / cm or less.

[22] The method according to

[21] above, wherein the eluting solution has an electrical conductivity of about 20 mS / cm to about 30 mS / cm.

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

[17] to

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

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

[17] to

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

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

[17] to

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

[26] The method according to

[26] above, wherein the CEX resin is operated in binding / elution mode.

[27] A method for producing a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The anti-α4β7 antibody is bound to the resin by contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin. Washing the mixed mode chromatography resin with a washing solution, The method includes obtaining a composition containing the anti-α4β7 antibody by contacting the resin with an elution solution having a pH of 4.2 or lower, thereby eluting the anti-α4β7 antibody from the mixed-mode chromatography resin. The method comprising the anti-α4β7 antibody, the heavy chain variable region sequence described in SEQ ID NO: 1 and the light chain variable region sequence described in SEQ ID NO: 5.

[28] The method according to

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

[29] The method according to

[27] or

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

[30] The method according to

[27] or

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

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

[27] to

[30] , wherein the eluting solution has an electrical conductivity of about 18 mS / cm to about 28 mS / cm.

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

[27] to

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

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

[27] to

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

[34] The method according to

[33] , wherein the mixed mode chromatography resin is brought into contact with approximately 55 g to approximately 80 g of the anti-α4β7 antibody per liter of resin.

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

[27] to

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

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

[27] to

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

[37] The method according to

[36] , wherein the CEX resin is operated in binding / elution mode.

[38] A method for producing a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The anti-α4β7 antibody is bound to the resin by contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a cation exchange (CEX) resin. The CEX resin is washed with a cleaning solution, The method involves contacting the resin with an elution solution having an conductivity of 16 mS / cm or less to elute the anti-α4β7 antibody from the CEX resin, thereby obtaining a composition containing the anti-α4β7 antibody. The method comprising the anti-α4β7 antibody, the heavy chain variable region sequence described in SEQ ID NO: 1 and the light chain variable region sequence described in SEQ ID NO: 5.

[39] The method according to

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

[40] The method according to

[38] or

[39] above, wherein the eluting solution has an electrical conductivity of 14 mS / cm or less.

[41] The method according to

[38] or

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

[42] The method according to

[38] or

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

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

[38] to

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

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

[38] to

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

[45] The method according to

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

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

[38] to

[45] , wherein the anti-α4β7 antibody is loaded into the CEX resin at a concentration of approximately 25 to 70 g of antibody per liter of resin.

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

[38] to

[46] , wherein the anti-α4β7 antibody is loaded into the CEX resin at a concentration of approximately 30 to 60 g of antibody per liter of resin.

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

[38] to

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

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

[38] to

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

[50] The method according to

[49] , wherein the mixed-mode chromatography resin is operated in binding / elution mode.

[51] A method for producing a composition containing an anti-α4β7 antibody from a liquid solution containing a major isoform of an anti-α4β7 antibody and one or more basic isoform species, The anti-α4β7 antibody is bound to the resin by contacting the liquid solution containing the anti-α4β7 antibody and one or more basic isoform species with a cation exchange (CEX) resin. The CEX resin is washed with a cleaning solution, The method involves contacting the resin with an elution solution having an conductivity of 11 mS / cm or higher to elute the anti-α4β7 antibody from the CEX resin, thereby obtaining a composition containing the anti-α4β7 antibody. The method comprising the anti-α4β7 antibody, the heavy chain variable region sequence described in SEQ ID NO: 1 and the light chain variable region sequence described in SEQ ID NO: 5.

[52] The method according to

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

[53] The method according to

[51] or

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

[54] The method according to

[51] or

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

[55] The method according to

[51] or

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

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

[51] to

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

[57] The method according to

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

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

[51] to

[57] , wherein the anti-α4β7 antibody is loaded into the CEX resin at a concentration of approximately 25 to 70 g of antibody per liter of resin.

[59] The method according to

[58] , wherein the anti-α4β7 antibody is loaded into the CEX resin at a concentration of approximately 30 to 60 g of antibody per liter of resin.

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

[51] to

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

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

[51] to

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

[62] The method according to

[61] above, wherein the mixed-mode chromatography resin is operated in binding / elution mode.

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

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

[64] The method according to

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

[65] The method according to any one of the above [1] to

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

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

[65] , comprising formulating the purified anti-α4β7 antibody as a dried lyophilized preparation.

[67] The method according to

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

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

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

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

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

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

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

[71] A composition comprising an anti-α4β7 antibody obtained by any of the methods described in [1] to

[70] above.

[0236] Sequence List [Table 10-1] [Table 10-2]

Claims

1. A method for obtaining a composition containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, The method involves obtaining a composition containing the anti-α4β7 antibody by contacting a solution containing the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-α4β7 antibody to flow through the HIC resin. The HIC resin is characterized as a highly hydrophobic HIC resin, and the HIC resin is equilibrated in a phosphate buffer containing about 0.15 M to about 0.35 M potassium phosphate and having a pH of less than about 7.

2. The aforementioned anti-α4β7 antibody was produced in Chinese hamster ovary (CHO) cells. The aforementioned anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and includes a heavy chain variable region containing the CDR3 domain described in SEQ ID NO: 4, the CDR2 domain described in SEQ ID NO: 3, and the CDR1 domain described in SEQ ID NO: 2, and includes a light chain variable region containing the CDR3 domain described in SEQ ID NO: 8, the CDR2 domain described in SEQ ID NO: 7, and the CDR1 domain described in SEQ ID NO:

6. The resulting composition containing the anti-α4β7 antibody contains less than 0.5% HMW aggregates. The aforementioned method.

2. The method according to claim 1, wherein the phosphate buffer has a pH of 5.5 to about 7.

3. The method according to claim 1 or 2, wherein the resin load is approximately 55 to 75 mg / ml.

4. The method according to any one of claims 1 to 3, wherein the composition contains less than about 0.22 ppm of residual protein A.

5. The method according to any one of claims 1 to 3, wherein the composition comprises less than about 0.3 ppm of host cell protein (HCP).

6. The method according to any one of claims 1 to 5, wherein the highly hydrophobic HIC resin has an average particle size of about 50 to 150 μm.

7. The method according to any one of claims 1 to 6, wherein the highly hydrophobic HIC resin has an average pore size of about 100 nm.

8. The method according to any one of claims 1 to 7, wherein the highly hydrophobic HIC resin has an average particle size of about 100 μm.

9. The method according to any one of claims 1 to 8, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence described in SEQ ID NO: 1 and the light chain variable region sequence described in SEQ ID NO:

5.

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

11. The method according to any one of claims 1 to 10, wherein the composition obtained comprises a purified anti-α4β7 antibody, and the method further comprises a subsequent step of formulating the anti-α4β7 antibody into a formulation suitable for human use.

12. The method according to any one of claims 1 to 11, wherein the anti-α4β7 antibody is vedolizumab.

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