Method for producing anti-α4β7 antibodies

Abstract

To provide a method for purifying an anti α4β7 antibody.SOLUTION: A method for purifying an anti α4β7 integrin antibody such as vedolizumab from liquid solution derived from a clarified harvest of a mammal cell culture, for example, includes: (i) a step of providing a harvest of a clarified cell culture obtained from a culture of a recombinant host cell which expresses the anti α4β7 antibody or an antigen-binding portion thereof; and (ii) a step of purifying the anti α4β7 antibody or an antigen-binding portion thereof from the harvest of the cell culture, where the antibody is exposed to pH of 4.0 or less for within 24 hours.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 859,494, filed on June 10, 2019. The entire content of the foregoing application is incorporated herein by reference.

[0003] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on June 5, 2020, is named T103022_1090WO_SL.txt and is 10,009 bytes in size.

Background Art

[0004] The large-scale and economical purification of proteins is becoming an increasingly important concern in the biotechnology industry. Generally, biopharmaceuticals are produced by cell culture using prokaryotes, such as bacteria, or eukaryotes, such as mammalian or fungal cell lines, designed to produce large amounts of the desired therapeutic protein. Since the cell lines used are living organisms, it is necessary to supply a complex cell culture medium containing sugars, amino acids, and growth factors, which may be supplied from preparations of animal serum. For example, cell culture medium components, host cell proteins (HCPs), host nucleic acids and / or chromatography materials, as well as product-related impurities such as aggregates, misfolded species, or fragments of the desired protein. [[ID=4l]] The isolation of desired recombinant therapeutic proteins from process-related impurities, including substances, is crucial for human therapeutics. Achieving a purity level sufficient for use as a therapeutic drug presents a significant challenge.

[0005] Product-related and process-related impurities, including aggregates, interfere with the purification process and storage. It may affect proteins, and / or administering antibodies to the target may cause side effects. This may be the cause of the effect (Shukla et al., J. Chromatog rBAnalyt.Technol.Biomed.Life Sci.,848( 1), 28-39).

[0006] Therefore, to effectively remove impurities, improve protein recovery rates, and meet therapeutic requirements While maintaining the need for technology to improve the purification of therapeutic proteins, such as antibodies, there is a need for such technology. It remains. [Overview of the project]

[0007] The present invention is at least in part based on the development of a process for producing an anti-α4β7 antibody or its antigen-binding moiety. In some embodiments, the anti-α4β7 antibody or its antigen-binding moiety is a humanized anti-α4β7 antibody or its antigen-binding moiety. In any of the following embodiments and aspects, the humanized anti-α4β7 antibody or its antigen-binding moiety may include a heavy chain variable region comprising CDR1 as described in SEQ ID NO: 2, CDR2 as described in SEQ ID NO: 3, and CDR3 as described in SEQ ID NO: 4 and / or a light chain variable region comprising CDR1 as described in SEQ ID NO: 6, CDR2 as described in SEQ ID NO: 7, and CDR3 as described in SEQ ID NO: 8. In some embodiments, the humanized anti-α4β7 antibody or its antigen-binding moiety includes a heavy chain variable region comprising SEQ ID NO: 1 and / or Sequence ID 5It includes a light chain variable region containing . In some embodiments, the humanized anti-α4β7 antibody, or its antigen-binding moiety, includes a heavy chain containing SEQ ID NO: 9 and / or a light chain containing SEQ ID NO: 10. In exemplary embodiments, the anti-α4β7 antibody is vedolizumab, or its antigen-binding moiety.

[0008] Therefore, in one embodiment, the present invention relates to a humanized anti-α4β7 antibody or its antigen-binding portion. This invention provides a method for producing a composition containing the following: pH greater than 6.5 Therefore, to provide a composition containing a humanized anti-α4β7 antibody or its antigen-binding moiety; humanized A composition containing an anti-α4β7 antibody or its antigen-binding moiety is administered for at least 20 minutes to 10 hours. To incubate; thereby containing the humanized anti-α4β7 antibody or its antigen-binding moiety To produce a composition.

[0009] In another aspect, the present invention relates to a humanized anti-α4β with reduced levels of basic isoform species. 7. A method for producing a composition containing an antibody or its antigen-binding moiety is provided, and this method is as follows: Includes: Humanized anti-α4β7 antibody, or a composition containing its antigen-binding moiety, with a pH exceeding 6.5 Provide at a certain pH; a composition containing a humanized anti-α4β7 antibody or its antigen-binding moiety. , the level of basic isoform species of the anti-α4β7 antibody or its antigen-binding moiety in the composition Incubate for a sufficient time to reduce the basic isophore; thereby, A humanized anti-α4β7 antibody with reduced levels of the α4β7 species, or a composition containing its antigen-binding moiety. To generate.

[0010] In some embodiments of the foregoing aspects, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising CDR1 set forth in SEQ ID NO: 2, CDR2 set forth in SEQ ID NO: 3, and CDR3 set forth in SEQ ID NO: 4 and / or a light chain variable region comprising CDR1 set forth in SEQ ID NO: 6, CDR2 set forth in SEQ ID NO: 7, and CDR3 set forth in SEQ ID NO: 8. In some embodiments, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising SEQ ID NO: 1 and / or Sequence ID 5 a light chain variable region comprising. In some embodiments, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain comprising SEQ ID NO: 9 and / or a light chain comprising SEQ ID NO: 10. In an exemplary embodiment, the anti-α4β7 antibody is vedolizumab, or an antigen-binding portion thereof.

[0011] In some embodiments, the method produces a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having < 16%, < 15%, < 14%, < 13%, < 12%, < 11% or < 10% basic isoform species.

[0012] In some embodiments, the incubation is performed during the purification of the humanized anti-α4β7 antibody or an antigen-binding portion thereof, and this incubation is (a) before ultrafiltration / diafiltration (UF / DF) of the antibody, or (b) before formulation of the antibody in a pharmaceutically acceptable buffer.

[0013] In some embodiments, this incubation is performed at ambient temperature. In some embodiments, this incubation is performed at 15 - 30 °C. In some embodiments, this incubation is performed at 20 - 25 °C.

[0014] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is provided at a pH of about 6.5 to 8.5. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is provided at a pH of about 7.0 to 8.0. In some embodiments, a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof is provided at a pH of about 7.0 to 7.5. In other embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is provided at a pH of about 6.6 to 7.3. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is at about pH 6 .5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, p H 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8 , pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, or pH 8.5.

[0015] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 10 to 120 hours. In some embodiments, a humanized anti-α4β7 antibody or a composition comprising an antigen-binding portion thereof is incubated for a period of about 10 to 120 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 96 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 72 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 48 hours. In some In some embodiments, the composition containing a humanized anti-α4β7 antibody or its antigen-binding moiety is small In some embodiments, humanized anti-α4 A composition containing a β7 antibody or its antigen-binding moiety was administered for at least 15 to 36 hours. It is incubated. In some embodiments, a humanized anti-α4β7 antibody or its antigen-binding agent is used. The composition containing the portion is incubated for a period of approximately 24 to 120 hours. Several implementations In terms of form, compositions containing a humanized anti-α4β7 antibody or its antigen-binding moiety are approximately 24-96 It is incubated for a period of time. In some embodiments, a humanized anti-α4β7 antibody is used. The composition containing the antigen-binding portion is incubated for approximately 24 to 72 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is It is incubated for a period of approximately 24 to 48 hours. In some embodiments, humanized anti-α4 Compositions containing β7 antibody or its antigen-binding moiety last for approximately 10 hours, approximately 12 hours, and approximately 24 hours. Incubation periods of approximately 36 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours. It will be done.

[0016] In another embodiment, provided herein are humanized anti-α4β7 antibodies or their antigens. This method involves purifying the binding portion from the harvested product of a clarified cell culture, and this method is (i ) Obtained from cultures of recombinant host cells expressing anti-α4β7 antibodies or their antigen-binding moieties. (ii) to provide harvested clarified cell cultures and (ii) an anti-α4β7 antibody or The antigen-binding portion is purified from the harvested cell culture, and this antibody is 4.0 or higher. When exposed to the following pH within 24 hours, this anti-α4β7 antibody or its antigen-binding moiety is distributed The heavy chain variable region containing the amino acid sequence described in column number 1, and the amino acid sequence described in Sequence ID No. 5 Includes a variable region of the light chain containing columns.

[0017] In some embodiments, the anti-α4β7 antibody or its antigen-binding moiety is compared to the control. The level of basic isoform species (determined by CEX) is reduced, and this The control is an anti-α4β7 antibody, or one containing its antigen-binding moiety, produced by the same method. This is a composition in which this antibody reacts to a pH of 4.0 or lower (for example, pH 3.6 to 4.0). Exposure for a longer period, i.e., more than 24 hours.

[0018] In some embodiments, the anti-α4β7 antibody, or its antigen-binding moiety, is vedolizumab or its antigen-binding portion.

[0019] In some embodiments, a humanized anti-α4β7 antibody, or its antigen-binding moiety, for example, Vedolizumab, or a composition containing its antigen-binding moiety, is a first basic isoform It includes a peak (BP1) and a second basic isoform peak (BP2), where this The method involves a humanized anti-α4β7 antibody or its antigen-binding moiety with reduced BP2 levels. A composition is produced. In a particular embodiment, this method produces a composition with less than 2%, less than 1.5%, and 1% Humanized anti-α4β7 antibodies, or their antigenicity, having BP2 less than or less than 0.7%. A composition containing the combined portion is produced.

[0020] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety. This is derived from mammalian cell cultures expressing humanized anti-α4β7 antibodies or their antigen-binding moieties. In certain embodiments, the mammalian cell culture is Chinese hamster ovary (CH O) Cell culture. In certain embodiments, the CHO cell culture is dihydrofolate redacted Includes CHO cells lacking tase (DHFR) expression. In certain embodiments, CHO cell culture The material includes CHO cells that lack glutamine synthetase (GS) expression.

[0021] In some embodiments, this method involves affinity chromatography, cation chromatography. Exchange chromatography, anion exchange chromatography, mixed-mode chromatography Ceramic hydroxyapatite (CHT) chromatography, and hydrophobic interaction One or more chromatographs selected from the group consisting of action chromatography (HIC). Using the isolation step, humanized anti-α4 is extracted from mammalian host cell proteins (HCPs). The method further comprises purifying a composition containing a β7 antibody or its antigen-binding moiety.

[0022] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is It is purified using an affinity chromatography resin containing protein A.

[0023] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is To reduce the basic isoform, affinity chloride is used before incubation. It is purified using matrixing. In certain embodiments, a humanized anti-α4β7 antibody is also used. The composition containing the antigen-binding moiety undergoes affinity chromatography after incubation. It is refined using Graph.

[0024] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is Purification is performed using cation exchange chromatography. In certain embodiments, humanized anti A composition containing an α4β7 antibody or its antigen-binding moiety was prepared before incubation. It is purified using exchange chromatography. In certain embodiments, humanized anti-α4β 7. Compositions containing an antibody or its antigen-binding moiety undergo cation exchange after incubation. It is purified using chromatography.

[0025] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is Purification is performed using anion exchange chromatography. In certain embodiments, humanized anti A composition containing an α4β7 antibody or its antigen-binding moiety was prepared before incubation. It is purified using exchange chromatography. In certain embodiments, humanized anti-α4β 7. Compositions containing an antibody or its antigen-binding moiety undergo anion exchange after incubation. It is purified using chromatography.

[0026] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety is Purification is performed using CHT chromatography. In certain embodiments, humanized anti-α4β 7. Compositions containing an antibody or its antigen-binding moiety are prepared by CHT chromatography before incubation. It is purified using tography. In certain embodiments, a humanized anti-α4β7 antibody or The composition containing the antigen-binding moiety is subjected to CHT chromatography after incubation. It is purified using [this method].

[0027] In some embodiments, this method includes incorporating the composition into a pharmaceutical formulation.

[0028] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. This freeze-dried pharmaceutical formulation is a dried, freeze-dried pharmaceutical formulation. In such an embodiment, the method is used to dry frozen so that it is suitable for administration. The process further includes the step of reconstituting the dried pharmaceutical formulation with a liquid.

[0029] In another embodiment, this pharmaceutical formulation is a liquid pharmaceutical formulation. In terms of administration methods, liquid pharmaceutical formulations are suitable for subcutaneous administration to humans.

[0030] In another embodiment, the present invention uses a humanized anti-α4β7 antibody or its antigen-binding moiety as a base This provides a method for reducing the level of sex isoform species and purifying this humanized anti-α4β7 The antibody or its antigen-binding portion is subjected to ultrafiltration / diafiltration (UF / DF). Therefore, in a pharmaceutically acceptable carrier, the humanized anti-α4β7 antibody or its antigen-binding portion is prepared. In response to the agent, the first harvesting of cell cultures yielded humanized anti-α4β7 antibody or its antigen-binding portion. At least 60%, at least 65%, at least 70%, of the total time until collection. At least 75%, at least 80%, at least 85%, at least 90%, at least pH 6 for 95%, at least 97%, at least 98%, or at least 99% Maintained at 0.5 or higher. In most of the purification process, humanized anti-α4β7 antibody or similar By maintaining the antigen-binding site at a pH of 6.5 or higher, the humanized anti-α4β7 antibody Alternatively, the basic isoform species of the antigen-binding portion can be used in an equivalent purification process. This is a humanized anti-α4β7 antibody or its antigen-binding portion, which is ultrafiltration / diafil tray By using (UF / DF), the harvested cell cultures are extracted from the pharmaceutically acceptable carrier. The t-transformed anti-α4β7 antibody or its antigen-binding portion is primarily recovered, and then the humanized anti-α4β7 antibody is also This refers to a significant portion of the total time until the antigen-binding portion is formulated, for example, more than 5%, 1 Between 0%, 15%, 20%, 25%, 30%, 35%, or 40%, p This can reduce the pH to less than H6.5 compared to an equivalent purification process.

[0031] In another aspect, the present invention relates to a humanized anti-α4β with reduced levels of basic isoform species. The present invention provides a composition comprising an antibody or its antigen-binding moiety (e.g., vedolizumab). In several embodiments, a humanized anti-α4β7 antibody, or a basic isopropyl alcohol of its antigen-binding moiety, is used. The form species is a humanized anti-α4β7 antibody present in the composition, or one of its antigen-binding moieties. Less than 5%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, Less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% Includes full. In some embodiments, the level of basic isoform species is reduced, human A composition comprising a chemical anti-α4β7 antibody or its antigen-binding moiety is prepared using the method described herein. It is produced by [method]. In some embodiments, the humanized anti-α4β7 antibody or its antigen-binding portion Compositions containing these components are produced by a combination of the methods of the present invention.

[0032] In another embodiment, provided herein is a method for producing a composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety (e.g., vedolizumab), the method comprising: (a) contact between a sample comprising a humanized anti-α4β7 antibody or its binding moiety antigen and a host cell protein (HCP) and an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a conductivity of 11 mS / cm or less, and the contact results in the HCP binding to the anion exchange resin; and (b) a flow-through material (flow through material) The method involves collecting a flow-through material from an anion exchange resin, the collection comprising a humanized anti-α4β7 antibody or its antigen-binding moiety.

[0033] In another embodiment, the present invention relates to a humanized anti-α4β7 antibody or its antigen-binding moiety (for example) The present invention provides a method for producing a composition comprising (a) humanized anti-α A sample containing a 4β7 antibody or its binding partial antigen and host cell protein (HCP) and an anonymity Contact with the on-exchange resin in the presence of a loading buffer, where this loading The buffer solution should be approximately 11 mS / cm or less (for example, approximately 11 mS / cm or less, approximately 10 mS / cm) Below, about 9mS / cm or less, about 8mS / cm or less, about 7mS / cm or less, about 6mS / cm Below are approximately 5 mS / cm or less, approximately 4 mS / cm or less, approximately 3 mS / cm or less, or approximately 2 mS It has an electrical conductivity of less than / cm, and as a result, HCP is bonded to the anion exchange resin. (b) bringing the anion exchange resin into contact with the elution buffer and (c) flow-through This involves collecting from an anion exchange resin, and this flow-through is humanized anti-α4β7 anti Includes collection of the body or its antigen-binding portion.

[0034] In some embodiments of the above-described model, this method involves a humanized anti-α4β7 antibody, or A composition is produced containing vedolizumab having an antigen-binding moiety, for example, a reduced amount of HCP. This method involves flow-through, which uses a humanized anti-α4β7 antibody, or its antigen-binding portion, for example. For example, vedolizumab and reduced amounts of HCP.

[0035] In some embodiments, the loading buffer is 9 mS / cm to 11 mS / cm (e.g.) For example, approximately 9 mS / cm to approximately 11 mS / cm, or approximately 10 mS / cm to approximately 11 mS / cm. It has the conductivity of ).

[0036] In some embodiments, the loading buffer is less than 11 mS / cm (e.g., 11 mS / cm). Less than mS / cm, less than 10 mS / cm, less than 9 mS / cm, less than 8 mS / cm, 7 mS / Less than cm, less than 6 mS / cm, less than 5 mS / cm, less than 4 mS / cm, less than 3 mS / cm It has an electrical conductivity of less than 2 mS / cm.

[0037] In some embodiments, the loading buffer is approximately 9 mS / cm, 9.5 mS / cm It has an electrical conductivity of 10 mS / cm, 10.5 mS / cm, or 11 mS / cm.

[0038] In some embodiments, HCP is Chinese hamster ovary (CHO) cell tannin. It is a protein. In certain embodiments, HCP is dihydrofolate reductase (DHFR) Derived from CHO cells lacking expression. In certain embodiments, HCP is glutamine synthetamine It is derived from CHO cells that lack GS expression.

[0039] In some embodiments, the anion exchange resin is washed with a washing buffer. In the embodiment, the washing buffer has a conductivity of 11 mS / cm or less. Several embodiments In this state, the washing buffer has a conductivity of 9 mS / cm to 11 mS / cm. Several implementations In some embodiments, the washing buffer has a conductivity of less than 11 mS / cm. The washing buffer is approximately 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS It has a conductivity of / cm or 11mS / cm. In some embodiments, the washing buffer has a conductivity of 11mS / cm. It has the same conductivity as the loading buffer.

[0040] In some embodiments, the loading buffer is sodium chloride and / or phosphoric acid. Contains sodium.

[0041] In some embodiments, the loading buffer is 20–150 mM salt, 50–12 It contains a 5 mM salt, or a 75-100 mM salt. In one embodiment, this salt is sodium chloride. Contains um and / or sodium phosphate. For example, buffer solutions are approximately 20 mM, approximately 30 mM. , about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about 10 0mM, approximately 110mM, approximately 120mM, approximately 130mM, approximately 140mM, or approximately 150m It may also contain M sodium chloride. Furthermore, or alternatively, the buffer may be about 20 mM. approx. 30mM, approx. 40mM, approx. 50mM, approx. 60mM, approx. 70mM, approx. 80mM, approx. 90m M, approximately 100mM, approximately 110mM, approximately 120mM, approximately 130mM, approximately 140mM, or It may contain approximately 150 mM sodium phosphate.

[0042] In some embodiments, the washing buffer is sodium chloride and / or sodium phosphate Includes mu.

[0043] In some embodiments, the washing buffer consists of 20–150 mM salt and 50–125 mM A salt, or a salt with a concentration of 75-100 mM. In one embodiment, this salt is sodium chloride and Contains and / or sodium phosphate. For example, buffer solutions of approximately 20 mM, approximately 30 mM, approximately 4 0mM, approximately 50mM, approximately 60mM, approximately 70mM, approximately 80mM, approximately 90mM, approximately 100mM Salt at approximately 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. It may also contain sodium hydroxide. Furthermore, or alternatively, the buffer may be about 20 mM, about 30 mM, approx. 40mM, approx. 50mM, approx. 60mM, approx. 70mM, approx. 80mM, approx. 90mM, approx. 100mM, approximately 110mM, approximately 120mM, approximately 130mM, approximately 140mM, or approximately 15 It may contain 0 mM sodium phosphate.

[0044] In some embodiments, the wash buffer has a conductivity equal to or greater than that of the loading buffer. It has low conductivity. In some embodiments, the washing buffer is the same as the loading buffer. It has the same conductivity.

[0045] In some embodiments, the anion exchange resin is used in an anion exchange column or anion exchange It is formatted as a replacement film.

[0046] In some embodiments, the anion exchange resin contains a quaternary amine functional group.

[0047] In some embodiments, a humanized anti-α4β7 antibody, or its antigen-binding moiety (for example, Samples containing vedolizumab and HCP are separated after one or more chromatographic separation steps. Derived from mammalian cell cultures. In certain embodiments, one or more chromatography The separation steps include affinity chromatography and cation exchange chromatography. Mixed-mode chromatography, hydrophobic interaction chromatography (HIC), and Selected from the group consisting of lamic hydroxyapatite (CHT) chromatography. Includes one or more steps.

[0048] In some embodiments, the amount of HCP in the flow-through is 8 ppm or less, or 7.5 ppm. m or less, 7ppm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm Below is 4.5 ppm. Below 4 ppm, below 3.5 ppm, below 3 ppm, below 2.5 ppm The concentration is below pm, or below 2 ppm.

[0049] In some embodiments, the amount of HCP in the flow-through is such that the conductivity is 12 mS / cm. This is generated when performing this method with the same sample using an excess loading buffer. For the amount of HCP in the flow-through, at least 50% (for example, at least approximately 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about (Decreases by 90%, at least about 95%, or at least about 98% or more than about 98%) .

[0050] In some embodiments, this method involves one or more pharmaceutically acceptable carriers or excipients. By a process including ultrafiltration and / or diafiltration into a buffer containing the agent This further involves processing the flow-through and replacing the buffer solution.

[0051] In some embodiments, this method includes incorporating the composition into a pharmaceutical formulation.

[0052] In certain embodiments, this pharmaceutical formulation is a lyophilized pharmaceutical formulation. In this context, freeze-dried pharmaceutical preparations are dry freeze-dried pharmaceutical preparations. In such embodiments, this method is used to dry and freeze-dry the material so that it is suitable for administration. The process further includes the step of reconstituting the prepared pharmaceutical formulation with a liquid.

[0053] In another embodiment, this pharmaceutical formulation is a liquid pharmaceutical formulation. In terms of administration methods, liquid pharmaceutical formulations are suitable for subcutaneous administration to humans.

[0054] In another aspect, provided herein are the results of any method of the present invention that generates A composition comprising a t-transformed anti-α4β7 antibody, or its antigen-binding moiety (e.g., vedolizumab) Yes, there are sets containing a humanized anti-α4β7 antibody or its antigen-binding moiety (e.g., vedolizumab). Products are also provided herein, and this composition is obtained by any method of the present invention. In these embodiments, the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, and 7 ppm. pm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5 Less than ppm, less than 4 ppm, less than 3.5 ppm, less than 3 ppm, less than 2.5 ppm, and The concentration is 2 ppm or less. In another embodiment, a humanized anti-α4β7 antibody or its antigen-binding agent is used. The basic isoform species of a portion (e.g., vedolizumab) is the antibody species present in the composition. Less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, 10% Less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, 2% Includes less than or less than 1%.

[0055] In some embodiments, a humanized anti-α4β7 antibody or its antigen-binding moiety (e.g., β) is used. Compositions containing dolizumab are produced by a combination of the methods of the present invention.

[0056] In another embodiment, the present invention provides recovery after elution from mixed-mode chromatography resins. The yield of the humanized anti-α4β7 antibody or its antigen-binding moiety (e.g., vedolizumab) Regarding the method of increasing the concentration, this method involves equilibrating the mixed-mode chromatography resin with a buffer solution. Equilibration is performed using a solution containing the anti-α4β7 antibody or its antigen-binding moiety and a low The dipping buffer is used to process the anti-α4β7 antibody or its antigen-binding portion, which is then chromatographed using mixed-mode chromatography. Loading the resin into a mixed-mode chromatography resin so that it bonds to the resin, and this The mixed mode chromatography resin is washed with washing buffer, and an anti-α4β7 antibody or anti- This includes eluting the original binding portion from the mixed-mode chromatography resin using an elution buffer. Here, the pH of the equilibration buffer, loading buffer, and / or washing buffer should be 7.0 or lower. It is below.

[0057] In one embodiment, the pH of the equilibration buffer, loading buffer, and / or wash buffer It is 6.0 to 7.0. In another embodiment, the equilibration buffer is a loading buffer and / or the pH of the washing buffer is 6.5-7.0. In another embodiment, the equilibration buffer is The pH of the loading buffer and / or washing buffer is 6.6 to 6.8.

[0058] In another embodiment, salts of the equilibration buffer, loading buffer, and / or wash buffer. The concentration is 30 mM to 70 mM. In another embodiment, equilibration buffer, loading buffer The salt concentration of the buffer and / or washing buffer is 40 mM to 70 mM. In another embodiment, The salt concentrations of the equilibration buffer, loading buffer, and / or wash buffer should be 50 mM. It is 65 mM. In another embodiment, the salt of the loading buffer and / or washing buffer. The concentration is 55 mM to 65 mM. In another embodiment, this salt is sodium chloride and / Or it contains sodium phosphate.

[0059] In another embodiment, salts of the equilibration buffer, loading buffer, and / or wash buffer. The sodium chloride concentration is 30 mM to 70 mM. In another embodiment, a loading buffer is used. The sodium chloride concentration of the solution and / or washing buffer is 40 mM to 70 mM. In this embodiment, sodium chloride is used in the equilibration buffer, loading buffer, and / or washing buffer. The lium concentration is 40 mM to 60 mM. In another embodiment, equilibration buffer, rhodochrosite The sodium chloride concentration of the rinsing buffer and / or washing buffer is 45 mM to 55 mM. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer are The sodium chloride concentration is approximately 50 mM. In another embodiment, equilibration buffer, Rhodi The washing buffer and / or the wash buffer further contain sodium phosphate.

[0060] In some embodiments, an equilibration buffer, a loading buffer, and / or a wash buffer are used. The pH of the solutions is the same. In some embodiments, equilibration buffer, loading buffer, The salt concentration of the and / or wash buffer is the same. In some embodiments, the equilibration buffer The loading buffer and / or washing buffer may be the same buffer. In some embodiments, the mixed mode resin is a ceramic hydroxyapatite resin. For example, CHT resin may also be used.

[0061] In another embodiment, provided herein is a low basic species composition containing an anti-α4β7 antibody. The substance is such that this composition contains less than 16%, less than 15%, less than 14%, or 1% of the anti-α4β7 antibody. Contains less than 3%, less than 12%, less than 11%, or less than 10% of total basic isoform species. Furthermore, this basic isoform species is positive when compared to the major isoform of the anti-α4β7 antibody. Possessing a positive charge, this anti-α4β7 antibody has a heavy chain variable region including SEQ ID NO: 1 and SEQ ID NO: The composition includes a light chain variable region containing 2. In some embodiments, a basic isopho The species can be quantified by cation exchange (CEX) chromatography. For example, In some embodiments, the basic isoform species corresponds to the primary isoform. The relative area of ​​the peak that elutes more slowly from the cation exchange (CEX) resin than the peak itself is determined. It can be quantified by determining its exact value.

[0062] Accordingly, in another embodiment, provided herein is a low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the total basic isoform species of the anti-α4β7 antibody, wherein the basic isoform species has a net positive charge compared to the major isoform of the anti-α4β7 antibody and can be quantified by determining the relative area of ​​the peak that elutes more slowly from the cation exchange (CEX) resin than the peak corresponding to the major isoform, and the anti-α4β7 antibody comprises a heavy chain variable region including SEQ ID NO: 1 and Sequence ID 5 The composition includes a light chain variable region containing [a specific element].

[0063] In some embodiments, this composition has a first basic isoform peak (BP1 ) and a second basic isoform peak (BP2) are included. In some embodiments, This composition contains less than 2% BP2. In some embodiments, this composition contains 1.5% It contains less than 1% BP2. In some embodiments, this composition contains less than 1% BP2. In some embodiments, this composition contains less than 0.7% BP2.

[0064] In some embodiments, the ratio of BP1 to BP2 is at least 3. In one embodiment, the ratio of BP1 to BP2 is at least 5. In some embodiments, the ratio of BP1 to BP2 is at least 7. The ratio of BP1 to BP2 is at least 10.

[0065] In some embodiments, this composition contains less than 8% total basic isoflavones of anti-α4β7 antibody. Contains foam species. In some embodiments, this composition contains less than 7% of the total anti-α4β7 antibody. It contains basic isoform species. In some embodiments, this composition contains anti-α4β7 antibodies It contains less than 6% of total basic isoform species. In some embodiments, this composition Contains less than 5% of the total basic isoform species of anti-α4β7 antibody.

[0066] In another embodiment, what is provided herein are compositions and pharmaceutically A pharmaceutical composition comprising an acceptable carrier or excipient. In some embodiments, this The pH of the pharmaceutical composition is between 6.0 and 7.0. In some embodiments, the pharmaceutical composition The pH of the product is approximately pH 6.3. In other embodiments, the pH of this pharmaceutical composition is p The temperature range is H6.3 to pH6.5.

[0067] In some embodiments, this pharmaceutical composition further comprises amino acids. In its applied form, this amino acid is either arginine or histidine.

[0068] In some embodiments, this pharmaceutical composition further comprises sugar. In certain embodiments, This sugar is either sucrose or trehalose.

[0069] In some embodiments, this pharmaceutical composition contains arginine, histidine, and sucrose. Contains -. In some embodiments, this pharmaceutical composition contains arginine, histidine, Contains sucrose and polysorbate 80.

[0070] In some embodiments, this pharmaceutical composition is at least 200 mg, at least 2 It contains 50 mg, or at least 300 mg, of anti-α4β7 antibody. In some embodiments, This pharmaceutical composition contains approximately 300 mg of anti-α4β7 antibody.

[0071] In some embodiments, the anti-α4β7 antibody is vedolizumab, or its antigen-binding moiety. That is the case.

[0072] In another embodiment, provided herein is a method for producing a low basic species composition comprising an anti-α4β7 antibody having a total basic isoform species content of less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%, the method comprising: providing a composition comprising the anti-α4β7 antibody at a pH higher than pH 6.3; incubating the composition comprising the anti-α4β7 antibody for a period of more than 10 hours; thereby producing a low basic species composition comprising the anti-α4β7 antibody having a total basic isoform species content of less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable region including SEQ ID NO: 1, and Sequence ID 5 Includes a light chain variable region.

[0073] In another embodiment, provided herein is a method for producing a low basic species composition comprising an anti-α4β7 antibody having a total basic isoform species content of less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%, the method comprising: providing a composition comprising vedolizumab at a pH higher than pH 6.3; incubating the vedolizumab composition for a time sufficient to reduce the level of basic vedolizumab isoform species in the composition; and thereby producing a low basic species composition comprising an anti-α4β7 antibody having a total basic isoform species content of less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable region including SEQ ID NO: 1, and Sequence ID 5 Includes a light chain variable region.

[0074] Generally, basic isoform species are compared to the main isoforms of anti-α4β7 antibodies. It has a net positive charge. In some embodiments, the level of the basic isoform species is It can be quantified using cation exchange chromatography (CEX). Several implementations In this state, the level of basic isoform species is lower than the peak corresponding to the major isoform. This method determines the relative area of ​​the peak that elutes more slowly from the cation exchange (CEX) resin. It can be quantified by the following. In some embodiments, this composition is a first basic iso It includes a form peak (BP1) and a second basic isoform peak (BP2). In some embodiments, this composition contains less than 2% BP2. This composition contains less than 1.5% BP2. In some embodiments, this composition contains 1 Contains less than % BP2. In some embodiments, this composition contains less than 0.7% BP2. include.

[0075] In some embodiments, the ratio of BP1 to BP2 is at least 3. In one embodiment, the ratio of BP1 to BP2 is at least 5. In some embodiments, the ratio of BP1 to BP2 is at least 7. The ratio of BP1 to BP2 is at least 10.

[0076] In some embodiments, this composition contains less than 8% total basic isoflavones of anti-α4β7 antibody. Contains foam species. In some embodiments, this composition contains less than 7% of the total anti-α4β7 antibody. It contains basic isoform species. In some embodiments, this composition contains anti-α4β7 antibodies It contains less than 6% of total basic isoform species. In some embodiments, this composition Contains less than 5% of the total basic isoform species of anti-α4β7 antibody.

[0077] In some embodiments, the following are provided herein: Antioxidants obtained by the aforementioned methods The composition contains an α4β7 antibody. In some embodiments, this antibody is vedolizumab or its antigen-binding portion.

[0078] In some embodiments, the following are provided herein: Antioxidants obtained by the aforementioned methods The composition contains an α4β7 antibody. In some embodiments, this antibody is vedolizumab or its antigen-binding portion.

[0079] In another embodiment, provided herein are methods for treating a disease or disorder in a human subject, the methods comprising administering to a subject a pharmaceutical composition provided herein, comprising an effective amount of an anti-α4β7 antibody (e.g., vedolizumab) for treating a disease or disorder in a human subject. In some embodiments, the pharmaceutical composition comprises a vedolizumab composition having reduced levels of basic vedolizumab isoform species and / or reduced levels of host cell proteins, produced as provided herein and / or according to the methods provided herein. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable region including SEQ ID NO: 1, and Sequence ID 5 Includes a light chain variable region.

[0080] In one embodiment, this disease or disorder is inflammatory bowel disease (IBD). In this embodiment, IBD is ulcerative colitis, Crohn's disease, ileitis, celiac disease, non-tropical Sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagen colitis, These include acidophilic gastroenteritis, pouchitis of the ileum following rectal and colectomy, and ileoanal anastomosis. In some embodiments, inflammatory bowel disease is Crohn's disease or ulcerative colitis. Additional diseases that may result include, for example, primary sclerosing cholangitis (PSC) and graft-versus-host disease. (GVHD) is one example.

[0081] Furthermore, the present invention also includes the following embodiments:

[0082] 1. Composition containing vedolizumab with reduced levels of basic vedolizumab isoform species. A method for generating an object, as follows: To provide a composition containing vedolizumab at a pH higher than pH 6.5; Incubate the vedolizumab-containing composition for more than 10 hours; As a result, vedolizumab with reduced levels of basic vedolizumab isoform species A method comprising producing a composition containing the above.

[0083] 2. The method according to item 1, wherein the incubation is performed at ambient temperature.

[0084] 3. The method according to item 1, wherein the incubation is performed at 15-30°C.

[0085] 4. The method according to item 1, wherein the incubation is performed at 20-25°C.

[0086] 5. The composition containing vedolizumab is provided at a pH of about 6.5 to 8.5, prior to The method described in any one of the items.

[0087] 6. The composition containing vedolizumab is provided at a pH of about 7.0 to 8.0, prior to The method described in any one of the items.

[0088] 7. The composition comprising vedolizumab is provided at a pH of about 7.0 to 7.5, prior to The method described in any one of the items.

[0089] 8. The composition containing vedolizumab has pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8. 1. The preceding item provided at pH 8.2, pH 8.3, pH 8.4, or pH 8.5 The method described in any one of the items.

[0090] 9. The composition containing vedolizumab is incubated for a period of approximately 10 to 120 hours. or the method described in any one of the preceding items.

[0091] 10. The composition containing vedolizumab is incubated for a period of approximately 12 to 120 hours. or the method described in any one of the preceding items.

[0092] 11. The composition containing vedolizumab is incubated for a period of approximately 12 to 96 hours. or the method described in any one of the preceding items.

[0093] 12. The composition containing vedolizumab is incubated for a period of approximately 12 to 72 hours. or the method described in any one of the preceding items.

[0094] 13. The composition containing vedolizumab is incubated for a period of approximately 12 to 48 hours. or the method described in any one of the preceding items.

[0095] 14. The composition containing vedolizumab is incubated for at least 12 hours. or the method described in any one of the preceding items.

[0096] 15. The composition containing vedolizumab is incubated for a period of approximately 24 to 120 hours. or the method described in any one of the preceding items.

[0097] 16. The composition containing vedolizumab is incubated for a period of approximately 24 to 96 hours. or the method described in any one of the preceding items.

[0098] 17. The composition containing vedolizumab is incubated for a period of approximately 24 to 72 hours. or the method described in any one of the preceding items.

[0099] 18. The composition containing vedolizumab is incubated for a period of approximately 24 to 48 hours. or the method described in any one of the preceding items.

[0100] 19. The composition containing vedolizumab was administered for approximately 12 hours, approximately 24 hours, approximately 36 hours, and approximately 4 hours. The preceding incubation period is 8 hours, approximately 72 hours, approximately 96 hours, or approximately 120 hours. The method described in any one of the items.

[0101] 20. The composition containing vedolizumab is used in mammalian cell cultures expressing vedolizumab. A method derived from any one of the preceding items.

[0102] 21. The mammalian cell culture is Chinese hamster ovary (CHO) cells. , the method described in item 20.

[0103] 22. The CHO cell culture lacks dihydrofolate reductase (DHFR) expression. The method described in item 21, including HO cells.

[0104] 23. The CHO cell culture lacks glutamine synthetase (GS) expression. The method described in item 21, including the cell.

[0105] 24. Affinity chromatography, cation exchange chromatography, anion Chromatography and ceramic hydroxyapatite (CHT) chromatography Using one or more chromatography separation steps selected from the group consisting of Raffy Therefore, a composition containing vedolizumab is purified from mammalian host cell proteins (HCPs). The method described in any one of the preceding items, further including the above.

[0106] 25. The composition containing vedolizumab is used in affinity chromatography containing protein A. The method described in item 24, which is purified using a grapheic resin.

[0107] 26. The composition containing vedolizumab has affinity prior to the incubation. - Purified using chromatography, as described in item 25.

[0108] 27. The composition containing vedolizumab exhibits affinity after incubation. The method described in item 25, which is purified using chromatography.

[0109] 28. The composition containing vedolizumab is purified by cation exchange chromatography. The method described in item 24.

[0110] 29. The composition containing vedolizumab undergoes cation exchange prior to the incubation. The method described in item 28, which is purified using chromatography.

[0111] 30. The composition containing vedolizumab undergoes cation exchange after the incubation. The method described in item 28, which is purified using chromatography.

[0112] 31. The composition comprising vedolizumab is obtained by anion exchange chromatography The method described in item 24 for purification.

[0113] 32. The composition containing vedolizumab undergoes anion exchange prior to the incubation. The method described in item 31, purified using chromatography.

[0114] 33. The composition containing vedolizumab undergoes anion exchange after the incubation. The method described in item 31, purified using chromatography.

[0115] 34. The composition containing vedolizumab is purified using CHT chromatography. The method described in item 24.

[0116] 35. The composition containing vedolizumab is prepared before the incubation using CHT chromatography. The method described in item 34, which is purified using tography.

[0117] 36. The composition containing vedolizumab undergoes CHT chromatography after the incubation. The method described in item 34, which is purified using tography.

[0118] 37. A composition comprising vedolizumab, the method described in any one of items 1 to 36. The composition produced by the above.

[0119] 38. The basic vedolizumab isoform species present in the composition The composition described in item 37, containing less than 10% of the mab species.

[0120] 39. Manufacture a composition containing vedolizumab in which the amount of host cell protein (HCP) is reduced. The method is as follows: (a) The sample containing vedolizumab and HCP was anionized in the presence of loading buffer. This involves bringing the replacement resin into contact with the load buffer, where the conductivity of the load buffer is HC P is bound to the anion exchange resin, and the contact is 11 mS / cm or less. to do; (b) Collecting flow-through material from the anion exchange resin, Here, the flow-through material contains vedolizumab and a reduced amount of HCP. The method, including collecting the above.

[0121] 40. The loading buffer has a conductivity of 9 mS / cm to 11 mS / cm. , the method described in item 39.

[0122] 41. The loading buffer has a conductivity of 10 mS / cm or less, item 39 Methods used.

[0123] 42. The loading buffer has a conductivity of 9 mS / cm or less, item 39 Methods used.

[0124] 43. The loading buffer is approximately 9 mS / cm, 9.5 mS / cm, and 10 mS / cm. Having an conductivity of 10.5 mS / cm or 11 mS / cm, as described in item 39. method.

[0125] 44. The HCP is Chinese hamster ovary (CHO) cell protein. The method described in any one of items 39 to 42.

[0126] 45. The aforementioned HCP is used in CHO cells that lack dihydrofolate reductase (DHFR) expression. The method described in item 44, which is derived from this method.

[0127] 46. ​​The HCPs mentioned above are derived from CHO cells lacking glutamine synthetase (GS) expression. The method described in item 44.

[0128] 47. Further comprising contacting the anion exchange resin with a washing buffer, item 39~ The method described in any one of item 46.

[0129] 48. The method according to item 47, wherein the washing buffer has a conductivity of less than 11 mS / cm .

[0130] 49. The washing buffer has a conductivity of 9 mS / cm to 11 mS / cm, item 4 The method described in 7.

[0131] 50. The washing buffer has the same conductivity as the loading buffer, as described in item 47. method.

[0132] 51. The loading buffer comprises sodium chloride and / or sodium phosphate. or the method described in any one of items 39 to 50.

[0133] 52. The washing buffer contains sodium chloride and / or sodium phosphate, item 3 The method described in any one of items 9 to 50.

[0134] 53. The anion exchange resin is used as an anion exchange column or anion exchange membrane. The method described in any one of items 39-52, which is matted.

[0135] 54. The anion exchange resin contains a quaternary amine functional group, any of items 39 to 53. The method described in item 1.

[0136] 55. Samples containing vedolizumab and HCP are detected in one or more chromatographic separation stages. The method described in any one of items 39-54, derived from mammalian cell cultures following the first step. .

[0137] 56. One or more chromatographic separation steps are performed in affinity chromatography. Cation exchange chromatography and ceramic hydroxyapatite (CHT Item 55 includes one or more steps selected from the group consisting of chromatography. Method of description.

[0138] 57. The amount of HCP in the eluate is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6. 5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less, 4 Less than ppm, less than 3.5 ppm, less than 3 ppm, less than 2.5 ppm, or less than 2 ppm The method described in any one of items 39 to 56.

[0139] 58. The amount of HCP in the flow-through material exceeds 12 mS / cm When the above method is performed using the same sample and a loading buffer, the following is generated The amount of HCP in the flow-through material is reduced by at least 50% compared to the item. The method described in any one of items 39 to 57.

[0140] 59. Ultrafiltration into a buffer containing one or more pharmaceutically acceptable carriers or excipients and / or to replace the elution buffer by a process that includes diafiltration Any one of items 39-58 further includes processing the aforementioned flow-through material. The method described in section [section number].

[0141] 60. A set containing vedolizumab produced by any one of items 39-59 Finished product.

[0142] 61. The amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, or 7 ppm or less. , 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less Below, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, or 2 ppm A composition described in item 60, which is less than or equal to m.

[0143] 62. Vedolizumab recovered after elution from mixed-mode chromatography resin A method for increasing the quantity, wherein the mixed mode chromatography resin is subjected to equilibration buffer. Equilibration, a solution containing vedolizumab and loading buffer, and the mixed mode Vedolizumab is loaded onto a chromatography resin and subjected to mixed-mode chromatography. To bind to the resin, the mixed mode chromatography resin is washed with a washing buffer. Washing and eluting the mixed-mode chromatography resin with elution buffer. This includes eluting the b, where the equilibration buffer, the loading buffer and / or The method wherein the pH of the washing buffer is 7.0 or less.

[0144] 63. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in item 62, wherein the pH is 6.0 to 7.0.

[0145] 64. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in item 62, wherein the pH is 6.5 to 7.0.

[0146] 65. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in item 62, where the pH is 6.6 to 6.8.

[0147] 66. The equilibration buffer, the loading buffer, and / or the washing buffer The method according to any one of items 62 to 65, wherein the salt concentration is 30 mM to 70 mM.

[0148] 67. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to item 66, wherein the salt concentration is 40 mM to 70 mM.

[0149] 68. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to item 66, wherein the salt concentration is 50 mM to 65 mM.

[0150] 69. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to item 66, wherein the salt concentration is 55 mM to 65 mM.

[0151] 70. The method according to any one of items 66 to 69, wherein the salt comprises sodium chloride and / or sodium phosphate. 9.

[0152] 71. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to any one of items 62 to 65, wherein the sodium chloride concentration is 30 mM to 70 mM. described method.

[0153] 72. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to item 71, wherein the sodium chloride concentration is 40 mM to 70 mM.

[0154] 73. For the equilibration buffer, the loading buffer, and / or the washing buffer The method according to item 71, wherein the sodium chloride concentration is 40 mM to 60 mM. <www

[0155] 74. For the equilibration buffer, the loading buffer, and / or the washing buffer The method described in item 71, wherein the sodium chloride concentration is 45 mM to 55 mM.

[0156] 75. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in item 71, wherein the sodium chloride concentration is approximately 50 mM.

[0157] 76. The equilibration buffer, the loading buffer, and / or the washing buffer Furthermore, the method described in any one of items 71 to 75, further comprising sodium phosphate.

[0158] 77. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in any one of items 62-76, wherein the pH is the same.

[0159] 78. The equilibration buffer, the loading buffer, and / or the washing buffer The method described in any one of items 62 to 77, wherein the salt concentration is the same.

[0160] 79. The equilibration buffer, the loading buffer, and / or the washing buffer The same buffer solution as described in any one of items 62-76.

[0161] 80. The mixed mode resin is a ceramic hydroxyapatite resin, item 62~ The method described in any one of item 79. [Brief explanation of the drawing]

[0162] [Figure 1] The cation exchange (CEX)-high-performance liquid chromatography (HPLC) profile of vedolizumab is shown, with peaks representing acidic species, basic species, and the major isoforms of vedolizumab. [Figure 2]The elution profiles of vedolizumab, purified using standard ceramic hydroxyapatite (CHT) equilibration and washing buffer after loading a CHT column with 27 mg / ml or 35 mg / ml of protein, are shown. [Figure 3] The elution profile of vedolizumab, purified using standard CHT equilibration and washing buffer or low pH buffer after loading 38 mg / ml of protein into a CHT column, is shown. [Modes for carrying out the invention]

[0163] This specification provides for anti-α4β7 integrin antibodies such as vedolizumab, for example. For example, a method for purifying liquid solutions derived from clarified harvests of mammalian cell cultures. Yes. In particular, the present invention relates to an anti-α4β7 integrin antibody, or its antigen-binding fragment. For example, product-related substances (e.g., bases) present in the purified preparation of vedolizumab. (e.g., acidic and / or acidic isoform species) and / or process-related impurities (e.g., stagnant Chief cell proteins (HCPs), host cell nucleic acids, viruses, chromatography materials, and This relates to a purification method for controlling the amount of (or culture medium components). Vedolizumab is relatively These are highly hydrophobic antibodies, and are particularly suitable for mass production at the purity level required for therapeutic applications. In some cases, there are challenges in purification.

[0164] I. Definition To make this invention easier to understand, certain terms are defined first.

[0165] Cell surface molecule "α4β7 integrin" or "α4β7" (used interchangeably throughout) It is a heterodimer of the α4 chain (CD49D, ITGA4) and the β7 chain (ITGB7). It is known. The human α4 integrin and β7 integrin genes GenBank (National Center for Biotechnology Information, B ethesda, Md.) respectively, RefSeq accession numbers NM_00088 5 and NM_000889) are expressed by B lymphocytes and T lymphocytes, particularly memory CD4+ lymphocytes. Typical of many integrins, α4β7 can exist in either a resting or activated state. Ligands for α4β7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., M AdCAM-1)). Antibodies that bind to α4β7 integrin are referred to herein as "anti- α4β7 antibodies".

[0166] As used herein, an antibody, or an antigen-binding fragment thereof, has "binding specificity for the α4β7 complex" which binds to α4β7 but does not bind to α4β1 or α B7. Vedolizumab is an example of an antibody having binding specificity for the α4β7 complex. E

[0167] The term "about" means that the value that follows is not an exact value but is the central point of a range of values of that value + / - 5%. When the value is a relative value expressed as a percentage, the term "about" means that the value that follows is not an exact value but is the central point of a range of values of that value + / - 5%, and the upper limit of that range cannot exceed a value of 100%. [[ID=3�]]

[0168] As used herein, the term "aggregate" or "aggregates" means two This refers to the association of one or more antibodies or antibody fragments. For example, an aggregate is an antibody and / or These were dimers, trimers, tetramers, or larger polymers than tetramers of antibody fragments. The antibody aggregates may be soluble or insoluble. The association between them was either covalent or non-covalent, regardless of the mechanism by which they met. It may also be an association between aggregated molecules, whether direct or otherwise linked together by other molecules. It may also be indirect, via molecules. Examples of the latter include, but are not limited to, other Disulfide bonding with proteins, hydrophobic association with lipids, charge association with DNA, leaching Examples include affinity association with tein A, or mixed association with multiple components. The aggregates are cells. Irreversibly during protein expression in culture, protein purification in downstream processing, or storage. Aggregates can be formed. The presence of aggregates in solution can be detected, for example, by size exclusion chromatography (SE). C) (For example, SEC with UV detection, SEC with light scattering detection (SEC-LSD)) ), field flow fractionation, analytical ultracentrifugation sedimentation velocity, or ca Pyrally electrophoresis - using sodium dodecyl sulfate (CE-SDS, reduced and unreduced) It can be decided by that.

[0169] As used herein, the term "antibody" refers to a group of bodies interconnected by disulfide bonds. An immunoglobulin consists of four polypeptide chains, two heavy (H) chains, and two light (L) chains. This refers to a phosphorus molecule. Each heavy chain has a heavy chain variable region (HCVR or VH in this specification). It consists of the heavy chain steady region (CH) and the heavy chain steady region (abbreviated as ). The heavy chain steady region consists of three domes It consists of CH1, CH2, and CH3. Each light chain has a light chain variable region (as specified herein). It consists of the LCVR (abbreviated as VL) and the light chain steady region. The domain consists of one domain CL. This VH and VL domain is a framework domain. Complementarity-determining regions (CDs) are scattered along with more conserved regions, known as FRs. It can be further subdivided into a region of ultra-variability called R). Each VH and VL is one of three CDs. It consists of R and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus. Reru: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Several actual In the application form, the antibody has a fragment crystallizable (Fc) region. In certain embodiments The antibody is of the IgG1 isotype and has a kappa light chain.

[0170] As used herein, the terms “basic species” or “basic isoform species” are defined as follows: This refers to a variant of an antibody or its antigen-binding portion, for example, vedolizumab, which is the whole thing. They are characterized by a basic charge. The basic species of the antibody or its antigen-binding site is positively charged. On-exchange high-performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or It can be detected by various methods known in the art, such as isoelectric focusing. Antibody salts The basal species are not limited to, but include charge variants, structural variants, and / Alternatively, fragmentation variants may be used. In some embodiments, antibodies Or, a composition containing the antigen-binding moiety contains two or more types of basic isoform species. It may be observed. In some embodiments, based on differences in retention times during CEX-HPLC separation Multiple basic isoform species can be identified. For example, antibodies, such as vedolizumab When analyzing the compositions containing these compounds using CEX-HPLC, as shown in Figure 1, each one or more basic isoform species of the antibody The species can be identified. For example, in some embodiments, the basic isoform species is aspergillus. This is an isoform of an antibody in which the laginate residue is isomerized to succinimide. Pure substances, or other impurities unrelated to the antibody or its antigen-binding portion, are determined by the primary sequence. , the "basic species" or "basic isoform species" of the antibody, or its antigen-binding portion and It is not considered.

[0171] As used herein, the term “buffer” refers to its acid-base conjugate A buffer solution is a solution that resists changes in pH due to the action of a certain substance. For example, a processing step or a chromatography resin or film such as a chromatographic It is used to establish biochemical conditions that mediate the control of the support material.

[0172] The "CDR" or "complementarity determination area" is referred to as the "framework area" (FR). , a region of hypervariability scattered within a more conserved region. The terms "antigen-binding fragment" or "antigen-binding portion" of an antibody are used by Fab, Fa b', F(ab')2, and Fv fragments, single-chain antibodies, functional heavy-chain antibodies (nanobody i) and at least one desired antibody that competes with intact antibodies for specific binding. Any part of an antibody that has specificity for the epitope (for example, specifically targeting the epitope) (Refers to an isolated portion of a complementarity-determining region that has a framework sequence sufficient for joining.) Antigen-binding fragments are obtained by recombinant technology, or by enzymatic or chemical processes of antibodies. It can be generated by cutting.

[0173] When used herein, "chromatographic support" refers to a support with a specific chemical composition. Or a specific three-dimensional solid or porous matrix, or a specific chemical group or Polymers are subjected to affinity chromatography and gel filtration (size exclusion chromatography). (i), or ion exchange chromatography, to perform chromatography This refers to a solid or porous matrix that can be fixed to a chromatography support. Examples, though not limited to them, include resins (e.g., agarose) or films. The term "chromatography housing" as used herein refers to a chromatography housing. This refers to a structure that includes a support. Examples of chromatography housings include columns or Examples include cartridges or other containers.

[0174] As used herein, the term “clarified harvest” refers to the protein of interest, for example For example, a liquid substance containing an anti-α4β7 antibody, which can be used in cell cultures, such as fermentation bioreactors. - After undergoing one or more process steps, the material contains solids such as cell fragments and particulate impurities derived from that substance. This refers to the liquid substance containing anti-α4β7 antibodies extracted after body particles have been removed. After cell culture, Typically, separation techniques such as centrifugation and filtration are used to purify the harvested material, and the cells and Remove cell debris. The initial clarification and particle removal step is, for example, a subsequent chromatography It yields a "clarified harvest" that can be used in the Raffi step (downstream processing). Processed harvested products are generally used as starting materials for downstream processing steps such as those described herein. It's about quality.

[0175] As used herein, the terms “culture” and “cell culture” generally refer to a culture of cells. This generally refers to (upstream) processes that grow outside of their natural environments under controlled conditions. To "culture" cells means to place them under conditions suitable for cell survival and / or growth and / or proliferation. This refers to bringing cells into contact with a cell culture medium. In certain embodiments, cell culture is intended to achieve a specific purpose. Recombinant proteins, for example, generate and maintain a population of host cells capable of producing anti-α4β7 antibodies. This refers to methods for maintaining, as well as methods and techniques for the production and collection of target proteins. For example, once an expression vector is incorporated into a suitable host, such as a host cell in culture... Then, the host expresses the relevant nucleotide coding sequence, as well as the desired recombinant protein. It can be maintained under conditions suitable for the collection and purification of the substance. "Cell culture" refers to a solution containing cells. It can also refer to this.

[0176] As used herein, the term “downstream process” refers to the protein of interest, for example. For example, it refers to one or more techniques used after an upstream process to purify antibodies. For example, downstream process technologies include affinity chromatography, and as an example, Rothene A affinity chromatography, anion or cation exchange chromatography Ion exchange chromatography such as Raffy, size exclusion chromatography, mixed chromatography Chromatic chromatography, hydrophobic interaction chromatography (HIC), or substitution chromatography This includes the purification of protein products using chromatography.

[0177] As used herein, the terms “elution solution” or “eluting solution” refer to the protein of interest. For example, replacing antibodies with a chromatography support, such as a resin or membrane. This refers to a formulated aqueous liquid. In one embodiment, the elution solution is a target protein, for example The antibody binds to the elution solution rather than to a chromatography support, such as a resin or membrane. Prioritizing the equilibration and / or washing solution, it has different biochemical properties from the equilibration and / or washing solution. .

[0178] As used herein, the term “equilibrium solution” refers to a processing step or chromatography. Fee support, for example, formulated to create initial operating conditions for chromatographic operations. It refers to an aqueous liquid. The equilibration solution is used to load the target protein, such as an antibody. For example, preparing a solid phase, for example, a chromatography support, for example, a resin or film. It is used for this purpose.

[0179] As used herein, "flow-through operation" refers to, for example, a chromatography step. In relation to this, impurities bind and remain bound to the chromatography support, This refers to the process by which proteins are eluted during loading and washing.

[0180] The term "high molecular weight" or "HMW" refers to antibodies with a molecular weight larger than that of monomer antibodies. It is used to represent an antibody complex. In one embodiment, the HMW aggregate is approximately 147 kD It has a molecular weight greater than a. The presence of high molecular weight aggregates is standard practice in the art. This can be determined by methods such as size exclusion chromatography (SEC).

[0181] The "humanized" form of non-human (e.g., rodent) antibodies is the minimal sequence derived from the non-human antibody. It is a chimeric antibody containing [something]. In most cases, humanized antibodies are superimposed on the recipient. Residues from the mutational region possess the desired specificity, affinity, and ability in mice, rats, and rabbits. or by residues from the hypervariable region of non-human species (donor antibodies) such as non-human primates. It is a replaceable human immunoglobulin (recipient antibody). In some cases, The framework region (FR) residues of the antibody are replaced with the corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either recipient or donor antibodies. These modifications are made to further improve antibody performance. Generally, humanized antibodies are less likely to be produced. It includes virtually all of the variable domains, at least one, typically two, and is a highly variable CDR. All or virtually all of the loops correspond to the hypervariable CDR loop of non-human antibodies, and all of the FRs Or, virtually all, are FRs of human antibody sequences. This humanized antibody is, at will, an anti At least a portion of the constant region (Fc) of the body, typically a small portion of the constant region (Fc) of a human antibody. This includes, but not all, parts of it. For further details, see, for example, Jones et al., N. ature 321:522-525(1986);Riechmann et al. , Nature 332:323-329(1988); and Presta, Curr. See Op. Struct. Biol. 2: 593-596 (1992).

[0182] The term "impurity" as used herein refers to impurities contained in a solution containing the purified antibody. The term "process-related" includes both process-related impurities and product-related impurities. As used herein, the term "impurity" refers to a composition containing, for example, protein. However, this refers to impurities (multiple impurities) present in the solution that do not originate from the protein itself. For example, process-related impurities include, but are not limited to, cell culture medium components and stagnant cells. Chief cell components (e.g., protein (HCP), host cell nucleic acid, or lipid-containing intracellular structures) (or fragments thereof), viruses, trace amounts of metal or ions derived from buffers, material processing containers or Examples include substances that can be leached from the chromatography support. Process-related impurities. These can be formed during the preparation of proteins, such as antibodies (upstream and / or downstream processing). As used herein, the term “host cell impurities” refers to host cell lines, cell cultures, etc. Protein, nucleic acid, and lipid contaminants introduced by liquid or cell culture, or refers to by-products. The term "host cell protein" refers to host cell lines, cell culture media, and This refers to protein by-products introduced through cell culture. Examples of impurities include limited It is not something that should be done, but Chinese hamster ovary protein (CHOP), E. col i protein, yeast protein, Simian COS protein, or myeloma cell protein Quality (for example, NS0 protein (mouse plasmacytoma cells derived from BALB / c mice)) It can be listed. Among host cell proteins, the target protein produced by the host cell expression system is Not included. For example, for CHO cells to produce recombinant antibodies or fragments thereof. When used, the term "host cell protein" refers to recombinant antibodies or their fragments. It includes proteins derived from CHO cells other than . The term "protein-related impurities" refers to impurities that originate from the target protein, such as the antibody itself. Includes, for example, product-related impurities, but not limited to, aggregation of the target antibody. Body, misfolded species, oxidized or deamidated species, or low molecular weight flags Mentions can be cited.

[0183] As used herein, the term “recombinant antibody” refers to a host cell, e.g., a mammal. Genes(s) that are held on recombinant expression vectors(s) introduced into a host cell(s) This refers to antibodies produced as a result of transcription and translation of recombinant proteins. In certain embodiments, recombinant proteins The proteins are IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, and IgA. 1. An isotype antibody selected from the group consisting of IgA2, IgD, or IgE. In certain embodiments, the recombinant antibody is IgG1.

[0184] The term "recombinant host cell" (in this specification, it is interchangeable with the term "host cell") (Used) includes cells into which recombinant expression vectors have been introduced. Such terminology is specific Please understand that this refers not only to the target cells but also to the descendants of such cells. A specific modification occurs in subsequent generations due to either mutation or environmental influences. In some cases, such offspring may not actually be identical to the parent cell. It remains within the scope of the term "host cell" as used herein. Furthermore, Unless otherwise specified, the term "cell" refers to, for example, a host cell or a host cell. It is understood that mammalian cells or mammalian host cells are intended to contain a population of cells. sea ​​bream.

[0185] "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%.

[0186] As used herein, "upstream" refers to proteins, for example, antibodies, and preparations. The term "process" refers to the production and collection of host cell-derived proteins (e.g., antibodies). This refers to activities that include (for example, during cell culture to produce a target protein, e.g., an antibody) (to).

[0187] As used herein, the term “vector” refers to a vector capable of growing another nucleic acid that is linked to it. This refers to nucleic acid molecules. This term includes vectors as self-replicating nucleic acid structures, as well as introduced into them. This includes vectors that are incorporated into the genome of host cells. Certain vectors are operational It can lead to the expression of nucleic acids linked to it. Such vectors are referred to herein as "expression vectors". To be called.

[0188] As used herein, the terms “cleaning” or “cleaning solution” refer to resins or membranes, etc. Aqueous liquid formulated to replace unbound contaminants from a chromatographic support. This refers to the following. In some embodiments, the washing material is loaded with the target protein, such as an antibody. Afterwards, and before eluting the target protein, such as an antibody, a solid support, such as a resin or It passes over the membrane. In one embodiment, the washing solution has biochemical properties similar to those of the equilibrium solution. .

[0189] II. Anti-α4β7 integrin antibody The method disclosed herein involves a highly purified mixture containing an anti-α4β7 integrin antibody. It can be used to mass-produce the product. As is clear, the anti-α4β described herein 7. Any method for producing integrin antibodies may be used individually or in combination. They may be used in combination. In exemplary embodiments, the antibody is vedolizumab or vedolizumab. It is an antibody that has one or more antigen-binding regions of zumab. Vedolizumab is also a trademarked product of ENT. YVIO (Registered Trademark) (Takeda Pharmaceuticals, Inc.) It is publicly known as: Vedolizumab is a human IgG1 framework and constant region, and Vedolizumab C is a humanized antibody containing an antigen-binding CDR derived from the mouse antibody Act-1. The DR, variable region, and mutated Fc region (mutated to eliminate Fc effector function) are, The entire text of U.S. Patent No. 7,147,851 is incorporated herein by reference. It is being done.

[0190] Vedolizumab specifically binds to α4β7 integrins, such as the α4β7 complex. It is a humanized monoclonal antibody. Vedolizumab is a metabolite that interacts with α4β7 integrin and mucosal adrenal receptors. It blocks the interaction with the cell adhesion molecule-1 (MAdCAM-1), causing inflammation. Vedolizumab inhibits the migration of memory T lymphocytes that have crossed the endothelium into the gastrointestinal parenchymal tissue. It can also bind to integrin α4β1 or αEβ7, or inhibit their function. In any case, the interaction between α4 integrin and vascular cell adhesion molecule-1 (VCAM-1) is antagonistic. I won't resist.

[0191] 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 intestinal lymphoid tissue. It plays an important role in the homing of T lymphocytes to α4β7 integrin and MAdCA. Interaction with M-1 is associated with mucositis, such as chronic inflammation, which is characteristic of ulcerative colitis and Crohn's disease. It is considered an important cause of the disease. Vedolizumab is used for inflammatory bowel disease, for example, in clots. This includes diseases such as ulcerative colitis, pouchitis, chronic pouchitis, graft-versus-host disease, and It may be used to treat HIV.

[0192] The heavy chain variable region of vedolizumab is provided herein as Sequence ID No. 1, and The vedolizumab light chain variable region is provided herein as Sequence ID No. 5. Mab contains CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4. It includes a heavy chain variable region. Vedolizumab is CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7 and includes a light chain variable region containing CDR3 of SEQ ID NO: 8. In one embodiment, this antibody is distributed It includes a heavy chain containing the amino acid sequence of sequence number 9, and a light chain containing the amino acid sequence of sequence number 10. Vedolizumab and the sequence of vedolizumab are also included in their entirety, and their entirety is referenced. More expressly incorporated herein are U.S. Patent Application Publication 2014 / 0341885 and This is also disclosed in U.S. Patent Application Publication No. 2014-0377251. The method involves using an antibody containing a binding region, for example, the CDR or variable region described above. You may go.

[0193] The method of the present invention involves using an anti-α4β7 antibody, particularly vedolizumab or vedolizumab, in mammalian cells. To produce an antibody that has a lizumab binding region, i.e., a CDR or variable region It is for use.

[0194] Exemplary strategies for antibody production In certain embodiments, the method described herein includes one or more steps described below. Includes one or more additional steps to facilitate the production and / or purification of vedolizumab. This can be performed in conjunction with the long-term, high-yield production of recombinant proteins such as vedolizumab. Therefore, mammalian host cells stably express an anti-α4β7 antibody (e.g., vedolizumab). It can be manipulated to do so. Exemplary cell culture processes, and monoclonal such as vedolizumab For a discussion on the production of Nal antibodies, see Li et al. (2010) mAbs 2:5,4 66-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685 (The full content is available by reference in this specification) It is described in (which is incorporated into).

[0195] In certain embodiments, primary recovery involves sequentially using the steps of pH reduction, centrifugation, and filtration. This method is used to remove cells and cell debris (including HCPs) from the harvested product of the production bioreactor. The process may be carried out by the following: In a particular embodiment, the present invention relates to the sample derived from the primary recovery. The mixture was analyzed using affinity chromatography, anion exchange (AEX), and cation exchange. (CEX), hydrophobic interaction chromatography (HIC), ceramic hydroxya Tight chromatography (CHT) and / or mixed mode (MM) purification steps It relates to providing one or more of these. In some embodiments, the order of steps is condensation By adjusting the levels of aggregates, impurities, or isoforms, the antibody composition can be modified. This can affect the quality of the resulting product.

[0196] In one exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Fee (for example, protein A), mixing mode, cation exchange, anion exchange in that order. It can be purified using a process that includes TEPS.

[0197] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., Protein A), mixed mode, cation exchange, anion exchange in that order. It can be purified using a process that includes steps.

[0198] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. The sequence is: Raffy (e.g., protein A), cation exchange, mixed mode, anion exchange. It can be purified using a process that includes steps.

[0199] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. The sequence is: Raffy (e.g., protein A), anion exchange, mixed mode, cation exchange. It can be purified using a process that includes steps.

[0200] In another exemplary embodiment, a composition comprising vedolizumab, mixing mode, affinity - Chromatography (e.g., protein A), anion exchange, cation exchange in that order. It can be purified using a process that includes steps.

[0201] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), CHT chromatography, cation exchange, anion It can be purified using a process that includes steps in the sequence of exchanges.

[0202] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), CHT chromatography, anion exchange, cation It can be purified using a process that includes steps in the sequence of exchanges.

[0203] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), cation exchange, CHT chromatography, anion It can be purified using a process that includes steps in the sequence of exchanges.

[0204] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), hydrophobic interaction chromatography, cation exchange It can be purified using a process that includes a sequence of anion exchange steps.

[0205] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), hydrophobic interaction chromatography, anion exchange It can be purified using a process that includes steps in a sequence of cation exchanges.

[0206] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), anion exchange, hydrophobic interaction chromatography It can be purified using a process that includes steps in a sequence of cation exchanges.

[0207] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), cation exchange, hydrophobic interaction chromatography It can be purified using a process that includes a sequence of anion exchange steps.

[0208] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), cation exchange, anion exchange, hydrophobic interaction chromosome It can be purified using a process that includes steps in the sequence of matography.

[0209] In another exemplary embodiment, a composition comprising vedolizumab is used in affinity chromatography. Raffy (e.g., protein A), anion exchange, cation exchange, hydrophobic interaction chromosome It can be purified using a process that includes steps in the sequence of matography.

[0210] In another exemplary embodiment, a composition comprising vedolizumab is used in hydrophobic interaction chromatography. Raffy, affinity chromatography (e.g., protein A), anion exchange It can be purified using a process that includes steps in a sequence of cation exchanges.

[0211] Please understand that the purification steps are not necessarily directly adjacent to each other. Filtration Other various process steps such as overcharging or virus reduction steps, charge profile Without disrupting the influence of the order of chromatography steps on the chromatograph, It may be inserted between steps.

[0212] To adjust the levels of basic isoform species present in the vedolizumab composition, As described herein, the incubation step is one of the purification steps described above. Any of these may be incorporated between them. Additionally or alternatively, the purification process is described herein. The antibody may be adapted to minimize the period during which it is exposed to low pH conditions. .

[0213] To regulate the levels of host cell proteins present in the vedolizumab composition, At any stage of the vedolizumab purification process using on-exchange chromatography, Honmei AEX may be performed using the low-conductivity buffer described in the detailed instructions.

[0214] To adjust the yield of vedolizumab in the purification process including CHT, this specification The CHT conditions described above are applied using ceramic hydroxyapatite chromatography. It can be used at any stage of the dolizumab purification process.

[0215] Certain embodiments of the present invention include a further purification step: ion exchange chromatography. Examples of additional purification steps that can be performed before, during, or after the process include ethanol precipitation. Isoelectric focusing, reverse-phase HPLC, silica chromatography, heparin Sephale Chromatography in S (trademark), further anion exchange chromatography and / or Further cation exchange chromatography, chromatographic focusing, SDS-PA GE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis , dialysis, and affinity chromatography (e.g., protein G, antibody, specific Examples include using a substrate, ligand, or antigen as a capture reagent.

[0216] In certain embodiments, the unbound flow-through and washing fractions may be further fractionated, targeting A pool of fractional combinations that provide the purity of the product may be used.

[0217] In certain embodiments, the protein concentration is adjusted to control the relationship between the antibody product and the product-related substance. Different distribution behaviors can be achieved, and as a result, purity and / or yield can be further improved. In one embodiment, loading is performed at different protein concentrations during the loading operation. It may be possible to improve the quality / yield of the product at any specific purification step.

[0218] In certain embodiments, to improve separation efficiency and / or yield of a particular purification step Alternatively, the column temperature may be changed independently.

[0219] In certain embodiments, the loading and washing solution matrix is ​​used for the novel separation described above. They may be different, while achieving similar "resin interaction" behavior, so that it can be achieved. Alternatively, it may consist of a mixture of chemical substances. For example, but not limited to loading The solution and the washing solution may differ in terms of ionic strength or pH, while the washing solution The functionality remains substantially similar in terms of washing out the products achieved during the step. Yes. In certain embodiments, additives such as amino acids, sugars, and PEG are added during the loading step. It can be added to the purging or washing step to adjust the separation behavior and achieve separation efficiency and / or yield. It is possible.

[0220] In certain embodiments, the loading and washing step is performed to improve the quality of the target product and / or The level of column discharge, collected pool, or both required to achieve the yield. In the product, the level of impurity / substance related to the product, inline, atline, or offline. It can be controlled by line measurement. In certain embodiments, the loading concentration is controlled by separation efficiency. and / or in order to achieve the distribution necessary to improve the yield, the solution or other solution It can be dynamically controlled by inline, batch, or serial dilution.

[0221] A particular embodiment of the present invention uses an ultrafiltration and diafiltration process, Further concentrate and formulate the target protein, such as an antibody product. Regarding ultrafiltration... , Microfiltration and Ultrafiltration: Pr principles and applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996) in; and Ultrafiltration Handbook, Munir Cheryan(Technomic Publishing,1986;ISBN N This is described in detail in o.87762-456-9). One of the filtration processes is "Ph armaceutical Process Filtration Catalog Millipore catalog titled "e", pages 177-202 (Bedford, Ma As described in ss., 1995 / 96), it is tangential flow filtration. Ultrafiltration generally refers to pore size This is thought to mean filtration using a filter smaller than 0.1 μm. By using a filter with a small pore size, proteins such as antibodies can be retained on the membrane surface. By allowing the sample solution to permeate the pores of the filter membrane, the volume of the sample can be reduced. ru.

[0222] Diafiltration uses a membrane filter to filter out salts, sugars, and non-aqueous solvents. Remove and replace, separate from bound species, remove low molecular weight species, and / or ions and / or a method that causes a rapid change in the pH environment. The minute solutes are affected by the flow rate of the permeate. The most effective method is to add the solvent to a solution that is diafiltration at an equal rate. They are removed at a rate. This washes away a certain amount of microscopic species from the solution, while retaining them. The target protein is effectively purified. In a particular embodiment of the present invention, diafil The purification step is optionally performed before chromatography or other purification steps. To replace various solutions used in connection with the invention, and to remove impurities from protein preparations. Used to remove objects.

[0223] Ultrafiltration / diafiltration (UF / DF) of vedolizumab involves equipment and membranes. For example, PELLICON® cassette (MilliporeSigma,Bu For example, polyethersulfone or regenerated cellulose (MMA), e.g. This can be done using either an ULTRACEL® or BIOMAX® membrane. In one embodiment, UF is a microporous polyethylene having a molecular weight cutoff of 30 kDa. This is performed using a cellulose membrane cast on a lens film.

[0224] III. Preparation of vedolizumab compositions containing basic isoform species with altered levels Vedolizumab preparations from mammalian host cells are usually major (also) In addition to the main vedolizumab isoform, there are also small amounts of acidic and / or This includes basic isoform species. Acidic and basic isoform species include, for example, cations. Known in the art, including CEX-HPLC. It can be quantified by method. Acidic and basic vedolizumab isoforms are CEX dendrocytes. They can be separated from the main isoforms based on differences in retention time in fat. Generally, Figure 1 As shown, the acidic vedolizumab isoform species present in the liquid preparation of vedolizumab It has a shorter retention time compared to the major isoforms, and is a basic vedolizumab isoform species. It has a longer retention time compared to the major isoform. Vedolizumab preparations are charged There are slight variations, resulting in multiple acidic species leaching from the CEX resin at different retention times. and / or may contain multiple basic species. Multiple basic peaks are retained on the CEX resin. Referenced herein in relation to time, following the major isoform peak, CEX resin or The first basic isoform peak that elutes is referred to herein as "basic peak 1". The second basic isoform elutes from the CEX resin following the main isoform peak. The foam peak is referred to herein as "basic peak 2" and is the main isoform peak. The third basic isoform peak eluting from the CEX resin following the peak is referred to herein as " This is sometimes called "basic peak 3".

[0225] In pharmaceutical antibody preparations, the percentage of antibodies present as the primary isoform. Maximize basic and / or acidic isoforms while minimizing the percentage of basic and / or acidic isoforms. It may be desirable to do so. In one aspect of the present invention, in a composition comprising vedolizumab This provides a method for adjusting the percentage of basic vedolizumab isoforms. The method shows that the distribution of vedolizumab isoforms can be regulated by changes in pH, which is quite remarkable. This is based on the discovery that the basic isoform of vedolizumab is particularly sensitive to pH fluctuations. As described herein, this pH-dependent regulation in isoform distribution. This is at least partially due to the variation in the level of basic peak 2, and the associated vedolizumab It is driven by variations in the levels of the major isoforms.

[0226] I do not wish to be bound by theory, but I believe that the findings presented herein are at least partially constrained. Based on this, at least two basic isoform species are present in some vedolizumab preparations. It is believed to exist. Firstly, it elutes from CEX resin as "basic peak 1". This is due to the presence of a lysine residue at the carboxyl terminus of the IgG heavy chain. Secondly, Elution from CEX resin as "basic peak 2" indicates the presence of one or more antibodies in asparagus. This is due to the isomerization of the acid residue to form a succinimide intermediate. Specific Embodiments Then, one or more aspartic acid residues undergo isomerization to form succinimide intermediates. The glycine or serine residue adjacent to aspartic acid is in the "n+1 position" (calcium carbonate). (Adjacent to the boxy terminus, nearly one amino acid) isoformation of the aspartic acid residue to succinimide. It can sometimes work to the advantage of chemical reactions. Succubi can be used instead of aspartic acid at residue 102 of SEQ ID NO: 1. The identification of this variant of vedolizumab containing niimide indicates that this basic isoform In some embodiments, the muvariant is reduced or minimized in the vedolizumab preparation. Alternatively, it becomes possible to remove it. In some embodiments, this basic isoform The variant is fractionated, for example, from a preparation containing vedolizumab (e.g., on CEX resin). This also involves the image of vedolizumab eluting from the CEX resin as "basic peak 2". It is also identifiable as a fraction, with residue 102 of SEQ ID NO: 1 having succinate instead of aspartic acid. By a method that includes removing (or being unable to collect) those fractions containing niimide It can be removed. In some embodiments, this basic isoform variant (in this specification) The book refers to it as "succinimide variant" or, alternatively, "basic isoform peak 2". The level of the antibody (also called the "BP2" variant) controls the pH exposure. This can be minimized during the production and purification of vedolizumab. In some embodiments, this Compositions with reduced levels of basic isoform variants, vedolizumab This may include a corresponding increase in the relative ratio of the isoforms. Therefore, several implementations In this state, a composition in which the level of this basic isoform variant is reduced, this basic Compared to compositions with increased levels of isoform variants, the potency may be enhanced.

[0227] In one embodiment, the present invention provides a reduction in the level of basic isoform species. This is a method for purifying vedolizumab, in which vedolizumab is purified by ultrafiltration / diaphragm Filtering (UF / DF) allows the cell culture to be transferred to a pharmaceutically acceptable carrier. Total time from the initial recovery of vedolizumab from the harvest to the formulation of vedolizumab. Of those, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, and Also 97%, at least 98%, or at least 99%, pH 5.5 or higher, pH 5. 6 or higher, pH5.7 or higher, pH5.8 or higher, pH5.9 or higher, pH6.0 or higher, pH6. Maintain a pH of 1 or higher, pH 6.2 or higher, pH 6.3 or higher, pH 6.4 or higher, or pH 6.5 or higher. Vedolizumab is maintained at a pH of 5.5 or higher for most of the purification process, and pH 5.6. Above, pH 5.7 or above, pH 5.8 or above, pH 5.9 or above, pH 6.0 or above, pH 6. Maintain a pH of 1 or higher, pH 6.2 or higher, pH 6.3 or higher, pH 6.4 or higher, or pH 6.5 or higher. By possessing this, the basic bendlizumab isoform species can be used in equivalent purification processes. In contrast, this can be reduced, and in this process, by ultrafiltration / diafiltration, After vedolizumab is first recovered from the harvest of cell cultures onto a pharmaceutically acceptable carrier, Significant time in the total time until vedolizumab is formulated, e.g., more than 5%, 10% Between over 15%, over 20%, over 25%, over 30%, over 35%, or over 40%, bed Lizumab is present at a pH of 5.5 to less than 6.5. In some embodiments, this method is used. For example, 1000L scale, 2000L scale, 3000L scale, 4000 Bed derived from harvested cell cultures produced on an L-scale or 5000L-scale Using lizumab preparations, this is carried out on a commercial manufacturing scale (e.g., at least 30 (00L scale).

[0228] Therefore, in one embodiment, the present invention relates to the low base of an anti-α4β7 antibody or its antigen-binding moiety. The present invention provides a method for producing a sex species composition, the method comprising (i) an anti-α4β7 antibody or its antigen. Harvesting of clarified cell cultures obtained from cultures of recombinant host cells expressing the binding site. (ii) To provide an anti-α4β7 antibody or its antigen-binding portion, in the collection of cell cultures This involves purification from the harvested material, where the antibody is at a pH of 3.5 or lower (for example, pH 2.5~). 3.5 (pH less than 3.0, or pH less than 3.5), for 20 minutes, 30 minutes, or 45 minutes. Within 1 hour, within 3 hours, within 5 hours, within 7 hours, within 10 hours, or 12 hours Within a certain period of exposure, this anti-α4β7 antibody or its antigen-binding moiety is more base-based than the control. The level of sex isoform species is reduced (determined by CEX), and this control However, it is a composition containing an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, This antibody is effective at pH levels below 3.5 (for example, pH 2.5 to 3.5, pH levels below 3.0, and (pH less than 3.5) for a long period of time, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, Exposure for more than 3 hours, more than 5 hours, more than 7 hours, more than 10 hours, or more than 12 hours. In this embodiment, the low basic species composition contains lower levels of BP2 compared to the control. In some embodiments, the antibody or its antigen-binding portion is the amino acid described in SEQ ID NO: 1. A heavy chain variable region containing a set of sequences, and a light chain variable region containing the amino acid sequence described in Sequence ID No. 5. Includes a region. In some embodiments, the antibody or its antigen-binding portion is vedolizumab, or the antigen-binding portion thereof. In some embodiments, an anti-α4β7 antibody, or the The step of purifying the antigen-binding site involves protein A chromatography and anion exchange chromatography. Chromatography, cation exchange chromatography, mixed-mode chromatography, This includes hydrophobic interaction chromatography and one or more combinations thereof. In some embodiments, host cells expressing an anti-α4β7 antibody or its antigen-binding moiety are These are GS-CHO cells. In other embodiments, the host cells are DHFR-CHO cells. In some embodiments, the low basic species composition contains less than 16%, less than 15%, and less than 14%. Basic isopropyl alcohols with a concentration of less than 13%, less than 12%, less than 11%, less than 10%, or less than 9%. Includes form species. In some embodiments, the composition of low basic species is less than 4%, 3% or less. Contains a basic isoform peak 2 at a concentration of less than 2%, or less than 1%.

[0229] In another embodiment, the present invention relates to a low basic species composition of an anti-α4β7 antibody or its antigen-binding moiety. The present invention provides a method for generating (i) an anti-α4β7 antibody or its antigen-binding portion We provide harvested clarified cell cultures obtained from cultures of recombinant host cells expressing the cell type. (ii) Extract the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture. This involves preparing the antibody at a pH of 4.0 or lower (for example, pH 3.6-4.0). Within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 3 hours, within 5 hours, 10:00 Exposure within 12 hours, 15 hours, 18 hours, or 24 hours, The anti-α4β7 antibody or its antigen-binding moiety is of a basic isoform species compared to the control. The level has decreased (determined by CEX), and this control was prepared in the same way. A composition comprising an anti-α4β7 antibody or its antigen-binding moiety, wherein this antibody is 4.0 or If the pH is below the normal range (for example, pH 3.6-4.0) for an extended period, i.e., more than 20 minutes, more than 30 minutes, 4 Over 5 minutes, over 1 hour, over 3 hours, over 5 hours, over 10 hours, over 12 hours, over 15 hours, 18:00 They are exposed for more than 24 hours. In some embodiments, the low basic species composition is Contains lower levels of BP2 compared to the control. In some embodiments, the antibody or The antigen-binding portion is a heavy chain variable region containing the set of amino acid sequences described in SEQ ID NO: 1, and It includes a light chain variable region containing the amino acid sequence described in SEQ ID NO: 5. In some embodiments, The antibody or its antigen-binding portion is vedolizumab, or its antigen-binding portion. In one embodiment, the step of purifying the anti-α4β7 antibody or its antigen-binding portion is performed. Rothene A chromatography, anion exchange chromatography, cation exchange chromatography Toography, mixed-mode chromatography, hydrophobic interaction chromatography, and and one or more of these combinations. In some embodiments, an anti-α4β7 antibody Alternatively, the host cells expressing the antigen-binding moiety are GS-CHO cells. Other embodiments The host cell is a DHFR-CHO cell. In some embodiments, a low basic species is used. The composition contains less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, and less than 11%. Containing a full, less than 10%, or less than 9% of basic isoform species. Several embodiments So, compositions with low basicity species, less than 4%, less than 3%, less than 2%, or less than 1% basicity Includes isoform peak 2.

[0230] In another embodiment, the present invention relates to a low basic species composition of an anti-α4β7 antibody or its antigen-binding moiety. The present invention provides a method for generating (i) an anti-α4β7 antibody or its antigen-binding portion We provide harvested clarified cell cultures obtained from cultures of recombinant host cells expressing the cell type. (ii) Extract the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture. This involves preparing the antibody, where the pH is 4.5 or lower (for example, pH 4.1-4.5). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, 24 hours Within 36 hours, within 48 hours, within 72 hours, within 96 hours, exposed to this anti-α The 4β7 antibody or its antigen-binding moiety showed a level of basic isoform species compared to the control. However, it has decreased (determined by CEX), and this control is an antibody produced in the same way. A composition containing an α4β7 antibody or its antigen-binding moiety, wherein the antibody has a p of 4.5 or less. For extended periods of time at H (e.g., pH 4.1-4.5), i.e., more than 3 hours, more than 5 hours, or 10 hours. Over, over 12 hours, over 18 hours, over 24 hours, over 36 hours, over 48 hours, over 72 hours, also It is exposed for a long period of 96 hours. In some embodiments, a low basic species composition is used as a control. It contains lower levels of BP2 compared to the antibody or its anti The primordial binding region includes a heavy chain variable region containing the amino acid sequence set described in Sequence ID No. 1, and the sequence It includes a light chain variable region containing the amino acid sequence described in number 5. In some embodiments, the antibody The antigen-binding portion is either vedolizumab or its antigen-binding portion. In the embodiment, the step of purifying the anti-α4β7 antibody or its antigen-binding portion is performed by Prote In-A chromatography, anion exchange chromatography, cation exchange chromatography Raffy, mixed-mode chromatography, hydrophobic interaction chromatography, and It includes one or more of these combinations. In some embodiments, it includes an anti-α4β7 antibody or The host cells that express the antigen-binding portion are GS-CHO cells. In other embodiments, The host cells are DHFR-CHO cells. In some embodiments, a set of low basic species For finished products, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, It contains less than 10% or less than 9% of basic isoform species. In some embodiments, low basic species composition, less than 4%, less than 3%, less than 2%, less than 1% basic isopho Includes Peak 2.

[0231] In another embodiment, the present invention relates to a low basic species composition of an anti-α4β7 antibody or its antigen-binding moiety. The present invention provides a method for generating (i) an anti-α4β7 antibody or its antigen-binding portion We provide harvested clarified cell cultures obtained from cultures of recombinant host cells expressing the cell type. (ii) Extract the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture. This involves preparing the antibody, where the pH is 5.0 or lower (for example, pH 4.6-5.0). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, 24 hours Within 36 hours, 48 ​​hours, 72 hours, or 96 hours of exposure, The anti-α4β7 antibody or its antigen-binding moiety is of a basic isoform species compared to the control. The level has decreased (determined by CEX), and this control was prepared in the same way. This composition contains an anti-α4β7 antibody or its antigen-binding moiety, and this antibody is 5.0 or If the pH is below the specified range (for example, pH 4.6-5.0) for an extended period, i.e., more than 3 hours, more than 5 hours, 1 Over 0 hours, over 12 hours, over 18 hours, over 24 hours, over 36 hours, over 48 hours, over 72 hours , or exposure for more than 96 hours. In some embodiments, the low basic species composition is a control. It contains lower levels of BP2 compared to the antibody or its anti The primordial binding region includes a heavy chain variable region containing the amino acid sequence set described in Sequence ID No. 1, and the sequence It includes a light chain variable region containing the amino acid sequence described in number 5. In some embodiments, the antibody The antigen-binding portion is either vedolizumab or its antigen-binding portion. In the embodiment, the step of purifying the anti-α4β7 antibody or its antigen-binding portion is performed by Prote In-A chromatography, anion exchange chromatography, cation exchange chromatography Raffy, mixed-mode chromatography, hydrophobic interaction chromatography, and It includes one or more of these combinations. In some embodiments, it includes an anti-α4β7 antibody or The host cells that express the antigen-binding portion are GS-CHO cells. In other embodiments, The host cells are DHFR-CHO cells. In some embodiments, a set of low basic species For finished products, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, It contains less than 10% or less than 9% of basic isoform species. In some embodiments, The low basic species composition contains less than 4%, less than 3%, less than 2%, or less than 1% basic i Includes Soform Peak 2.

[0232] In another embodiment, the present invention relates to a low basic species composition of an anti-α4β7 antibody or its antigen-binding moiety. The present invention provides a method for generating (i) an anti-α4β7 antibody or its antigen-binding portion We provide harvested clarified cell cultures obtained from cultures of recombinant host cells expressing the cell type. (ii) Extract the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture. This involves preparing the antibody, where the pH is 5.5 or lower (for example, pH 5.1-5.5). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, 24 hours Within 36 hours, 48 ​​hours, 72 hours, or 96 hours of exposure, The anti-α4β7 antibody or its antigen-binding moiety is of a basic isoform species compared to the control. The level has decreased (determined by CEX), and this control was prepared in the same way. This composition contains an anti-α4β7 antibody or its antigen-binding moiety, and this antibody is 5.5 or If the pH is below the specified range (for example, pH 5.1-5.5) for an extended period, i.e., more than 3 hours, more than 5 hours, 1 Over 0 hours, over 12 hours, over 18 hours, over 24 hours, over 36 hours, over 48 hours, over 72 hours , or exposure for more than 96 hours. In some embodiments, the low basic species composition is a control. It contains lower levels of BP2 compared to the antibody or its anti The primordial binding region includes a heavy chain variable region containing the amino acid sequence set described in Sequence ID No. 1, and the sequence It includes a light chain variable region containing the amino acid sequence described in number 5. In some embodiments, the antibody The antigen-binding portion is either vedolizumab or its antigen-binding portion. In the embodiment, the step of purifying the anti-α4β7 antibody or its antigen-binding portion is performed by Prote In-A chromatography, anion exchange chromatography, cation exchange chromatography Raffy, mixed-mode chromatography, hydrophobic interaction chromatography, and It includes one or more of these combinations. In some embodiments, it includes an anti-α4β7 antibody or The host cells that express the antigen-binding portion are GS-CHO cells. In other embodiments, The host cells are DHFR-CHO cells. In some embodiments, a set of low basic species For finished products, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, It contains less than 10% or less than 9% of basic isoform species. In some embodiments, The low basic species composition contains less than 4%, less than 3%, less than 2%, or less than 1% basic i Includes Soform Peak 2.

[0233] In some embodiments, this method can be used for, for example, 1000L scales, 2000L scales. 3000L scale, 4000L scale, or 5000L scale (for example, Vedolizma derived from harvested cell cultures produced on a scale of at least 3000L This is carried out on a commercial production scale using the preparations of [product name].

[0234] In some embodiments, the methods described herein include compositions, for example, vedolizumab. Used to adjust the level of basic vedolizumab isoforms in liquid compositions containing them. In some embodiments, this method can be used to obtain low levels of basic vedolizumab. A vedolizumab composition containing seeds may be prepared.

[0235] In one embodiment, the present invention reduces the level of basic vedolizumab isoform species. By incubating the composition at a pH above 6.5 for a sufficient amount of time, the bedrid A method for reducing the level of basic vedolizumab isoform species in a zumab-containing composition. This provides. In one embodiment, this method is carried out at an ambient temperature, for example, 20-25°C. In other embodiments, this method is carried out at 2-8°C.

[0236] In another embodiment, the present invention relates to a vedolizumab isoform species with reduced levels. The present invention provides a method for producing a composition containing dolizumab. This method requires a pH of 6.5 or higher. To provide a composition containing vedolizumab, and to provide a composition containing vedolizumab with basic vedolizumab Incubate for a sufficient amount of time to reduce the level of rizumab isoform species. Furthermore, it includes vedolizumab with reduced levels of basic vedolizumab isoform species. This may include generating a composition.

[0237] Efficiently reduces the level of basic vedolizumab isoform species in vedolizumab compositions. To achieve this, incubation is preferably carried out at a pH of 6.5 or higher.

[0238] In exemplary embodiments, the pH of the vedolizumab composition is approximately pH 6.5 to 9.0. For example, the pH of the vedolizumab composition is approximately pH 6.5-9.0, pH 6.5-8.5, p The temperature may be between H6.5 and 8.0, and the pH may be between 6.5 and 7.5, or within the range of pH 6.5 and 7.0. Alternatively, the pH of the vedolizumab composition is approximately pH 7.0-9.0, pH 7.5-9.0. The pH may be in the range of 8.0 to 9.0, or 8.5 to 9.0. In other embodiments, The pH of the vedolizumab composition is approximately 6.5 to 7.5, for example, pH 6.6 to 7.3. pH6.6~7.5, pH6.7~7.5, pH6.8~7.5, pH6.9~7.5, pH7.0~7.5, pH7.1~7.5, pH7.2~7.5, pH7.3~7.5, Or it may be in the range of pH 7.4 to 7.5. In other embodiments, the vedolizumab composition The pH range is approximately pH 6.5-7.5, pH 6.5-7.4, pH 6.5-7.3, and pH 6.5. ~7.2, pH6.5~7.1, pH6.5~7.0, pH6.5~6.9, pH6.5 It can be in the range of ~6.8, pH 6.5~6.75, or pH 6.5~6.6. Other actual In the application form, the pH of the vedolizumab composition may be in the range of approximately 7.0 to 7.5. In the application form, the pH of the vedolizumab composition may be in the range of approximately 7.5 to 8.0. In the application configuration, the pH of the vedolizumab composition may be in the range of approximately 8.0 to 8.5. (Example) In one embodiment, the pH of the vedolizumab composition is approximately pH 6.5, pH 6.6, and pH 6.7. , pH6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7 .4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, p H8.1, pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7 The pH may be 8.8, 8.9, or 9.0.

[0239] A composition containing vedolizumab at the above pH, for example, a pH of 6.5 or higher, vedolizumab Sufficient to reduce the percentage of basic vedolizumab isoform species in the composition It can be incubated for a certain amount of time. In an exemplary embodiment, incubation is 20 minutes or less. Above, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hours or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 1 8 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, 120 hours or more The above is carried out over a period of 144 hours or more, or 168 hours or more. Several embodiments So, incubation is approximately 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or less The above is carried out for a period of 5 days or more, 6 days or more, or 7 days or more. In some embodiments, the ink The introductory sessions are approximately 20 minutes to 1 hour, 20 minutes to 1 hour, 20 minutes to 2 hours, and 2 hours. 0 minutes to approximately 3 hours, approximately 1 hour to approximately 3 hours, approximately 1 hour to approximately 5 hours, or approximately 5 hours to approximately 8 hours It is permissible to do so. In some embodiments, incubation is from about 8 hours to about 168 hours. It may be carried out for more than 7 hours. In some embodiments, incubation The timeframe is approximately 8 to 168 hours, for example, approximately 8 to 144 hours (6 days), approximately 8 to 120 hours (5 days) ), approximately 8-96 hours (4 days), approximately 8-72 hours (3 days), approximately 8-48 hours (2 days), approximately 8 ~36 hours, approximately 8-24 hours (1 day), approximately 8-18 hours, approximately 8-12 hours, approximately 15-36 hours It may be carried out for 1 hour, or about 8-10 hours. In other embodiments, incubation is Approximately 10 hours to over 168 hours, for example, approximately 10-168 hours, approximately 12-168 hours, approximately 18-168 hours, approximately 24-168 hours, approximately 36-168 hours, approximately 48-168 hours Approximately 72-168 hours, approximately 96-168 hours, approximately 120-168 hours, or approximately 144- It may be carried out for 168 hours. In some embodiments, incubation is about 0.5~ For example, 7 days, approximately 0.5-5 days, approximately 0.5-4 days, approximately 0.5-3 days, approximately 0.5-2 days, Alternatively, it may be carried out for about 0.5 to 1 day. In some embodiments, incubation is This may be done for approximately 1 to 5 days, approximately 1 to 3 days, or approximately 1 to 2 days. In an exemplary embodiment, p Incubation at H6.5 or higher is for a period of 1-2 days, 1-3 days, or 1-5 days. This is done over time. In other exemplary embodiments, incubation at pH 6.5 or higher is performed. Total purification time, for example, starting from the provision of harvested clarified cell cultures and the purified antibody This is done over more than 25% of the time the body is in the UF / DF phase.

[0240] In other embodiments, incubation involves the basic vedolizumab isophore in the composition. The percentage of the room should be 1% or more, for example, 1.5% or more, 2% or more, 2.5% or more. 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 6% or more, 7% or more, 8 % or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 1 5% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% This is carried out for a period of time sufficient to reduce by more than 100% or more. In an exemplary embodiment, Incubation involves determining the percentage of basic vedolizumab isoform species in the composition. This is done for a period of time sufficient to reduce the dose by 1% to 5%. In other embodiments, incubation The percentage of basic vedolizumab isoform species in the composition is 1% to 10%. This is carried out for a sufficient period to reduce by %. In other embodiments, incubation is performed on the composition Reduces the percentage of basic vedolizumab isoform species in a substance by 2% to 10%. This is carried out for a sufficient period of time. In other embodiments, incubation is performed to determine the basicity of the composition. A sufficient period to reduce the percentage of vedolizumab isoform species by 5% to 10% This is carried out over time. In other embodiments, incubation is performed on the basic vedolizumab in the composition. This should be done for a sufficient period to reduce the percentage of isoform species by 2% to 20%. In other embodiments, incubation involves the basic vedolizumab isophor in the composition. This should be done for a sufficient period to reduce the percentage of the M species by 5% to 20%.

[0241] In other embodiments, incubation involves an anti-α4β7 antibody (e.g., vedolizumab). This process is carried out for a sufficient period of time to produce a low basic species composition containing less than 16%. Less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, or 9 It has less than % total basic isoform species. In some embodiments, this method is used for salt The level of the base isoform peak 2 is less than 4%, less than 3%, less than 2%, or less than 1%. Incubate the composition at a pH above 6.3 for a sufficient amount of time to reach fullness. This includes. In some embodiments, this method involves preparing a composition containing vedolizumab at pH 6. pH levels higher than 3 (for example, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6) .8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, p H7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1 , pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7, pH8 Incubate at 0.8, pH 8.9, or pH 9.0; use an anti-α4β7 antibody (e.g., For example, a composition containing vedolizumab) for 20 minutes or more, for example, 30 minutes or more, 1 hour or more, 2 Over 1 hour, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more , 10 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 24 hours or more, 48 hours The above applies to 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours. This includes incubation for more than two hours.

[0242] During incubation, in some embodiments, the pH of the vedolizumab composition is pH 6.3 or higher can be maintained. During incubation, in other embodiments, the vedolizumab compound The pH of the product can be maintained at pH 6.5 or higher. In some embodiments, vedolizumab The composition is maintained at approximately the same pH throughout the incubation period.

[0243] Incubation can be carried out at any suitable temperature. For example, incubation The process can be carried out at temperatures in the range of approximately 0 to 40°C. In another embodiment, incubation The process can be carried out at temperatures in the range of approximately 1 to 37°C. In some embodiments, the incubation The procedure is carried out at temperatures in the range of approximately 0-4°C or 4-8°C. Other embodiments So, this incubation is carried out at ambient temperature. For example, incubation is, This can be carried out at temperatures in the range of approximately 20-25°C. In other embodiments, incubation is The incubation is carried out at approximately 37°C. In some embodiments, the incubation is performed at approximately 1 to 25°C. The incubation is carried out at temperatures in the range of °C. In some embodiments, the incubation is about 5°C The incubation is carried out at temperatures in the range of 18°C. In some embodiments, incubation is approximately The process is carried out at temperatures in the range of 15-30°C. In some embodiments, incubation This is carried out at a temperature in the range of approximately 33-37°C. In an exemplary embodiment, incubation The temperature ranges are 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃ ℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21 ℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31 ℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40 It is performed in °C.

[0244] The method described above optionally includes an additional step of adjusting the pH of the vedolizumab composition. That is also acceptable. For example, the percentage of basic vedolizumab species in a composition containing vedolizumab If it is desirable to reduce the amount of p of the composition before incubation, this method is used. The step may include increasing H. For example, if the composition containing vedolizumab has a H of less than 6.5. If the pH is 6.5, this method includes the step of raising the pH of the composition to 6.5 or higher. But that's fine too. Conversely, the percentage of basic vedolizumab species in a composition containing vedolizumab If it is desirable to increase the pH, this method can be used to adjust the pH of the composition before incubation. The step may include lowering the pH. For example, the composition containing vedolizumab has a pH of 6.5 or lower. If the condition is met, this method may include a step of lowering the pH of the composition to less than 6.5. good.

[0245] The composition containing vedolizumab may be a liquid solution. In some embodiments, Compositions containing dolizumab are used to produce vedolizumab or its antigen-binding moiety. It originates from the host cell being used. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells, e.g. For example, CHO cells lacking dihydrofolate reductase (DHFR) expression, or glutamine These are CHO cells lacking synthetase (GS) expression.

[0246] The aforementioned method involves small or large-scale vedolizumab purification following the isolation of antibodies from cell culture. It may be incorporated into the simulation process. In certain embodiments, vedolizma from host cell cultures Primary recovery of spores, for example, bioreactor harvesting, uses the steps of centrifugation and filtration. The method described herein may be performed before or after centrifugation and / or before filtration. Later, the vedolizumab composition is subjected to the corresponding purification step of the base in the composition. The level of sexual isoform species can be reduced. After primary recovery, process-related impurities and / or or downstream process steps that can be used to purify vedolizumab from product-related impurities. While not limited to this, affinity chromatography (protein A Chromatography, deep filtration, cation exchange (CEX), anion exchange (AEX) ), mixed-mode chromatography (MM), ceramic hydroxyapatite chromatography Chromatography (CHT), hydrophobic interaction chromatography (HIC), ultrafiltration, and / or diafiltration may be used. The methods described herein are downstream processes. Perform this on the vedolizumab composition before or after the step, and in the corresponding step of purification, the composition This can reduce the level of basic isoform species within the molecule.

[0247] For example, the vedolizumab composition is used before or after affinity chromatography. It can be incubated as described herein. In one embodiment, this method The method involves vedolizumab compositions, for example, affinity chromatography load materials. Set the pH of the eluate from the eluate or affinity chromatography to pH 6.5 or higher. This may include making adjustments to it.

[0248] In another embodiment, the vedolizumab composition is used before or after deep filtration as described herein. It can be incubated to a pH of 6.5 or higher. In one embodiment, this method can be incubated to a pH of 6.5 or higher. This may include adjusting the pH of the vedolizumab composition before or after deep filtration. .

[0249] In another embodiment, the vedolizumab composition is used before or after cation exchange (CEX). It can be incubated as described herein. In one embodiment, this method is used The pH of the dolizumab composition, for example, CEX load material, or CEX eluate, is set to p This may include adjusting the pH to H6.5 or higher.

[0250] In another embodiment, the vedolizumab composition is used before or after anion exchange (AEX). The incubation may be carried out as described herein. In one embodiment, this method , vedolizumab composition, for example, AEX load material, or AEX flowthrough This may include adjusting the pH to a pH of 6.5 or higher.

[0251] In another embodiment, the vedolizumab composition is subjected to mixed-mode chromatography (MM) Incubation may be performed before or after as described herein. In one embodiment, This method involves a vedolizumab composition, for example, MM load material, or MM eluate. This may include adjusting the pH to a pH of 6.5 or higher.

[0252] In another embodiment, the vedolizumab composition is a ceramic hydroxyapatite chromatograph. Incubate before or after the Graph (CHT) as described herein. This is also acceptable. In one embodiment, this method involves a vedolizumab composition, for example, CHT load mate. This may include adjusting the pH of the real or CHT eluate to a pH of 6.5 or higher. good.

[0253] In another embodiment, the vedolizumab composition is subjected to hydrophobic interaction chromatography (HI It may be incubated before or after C) as described herein. One Embodiment So, this method involves a vedolizumab composition, for example, HIC load material, or HI This may include adjusting the pH of the C eluate to a pH of 6.5 or higher.

[0254] In another embodiment, the vedolizumab composition is ultrafiltered and / or diafil tray Incubate before or after the (UF / DF) as described herein. Alternatively, in one embodiment, this method involves adjusting the pH of the vedolizumab composition before UF / DF. This may later include adjusting the pH to 6.5 or higher.

[0255] IV. Vedolizumab compositions with reduced levels of basic isoform species. In some embodiments, the present invention relates to low-level basic vedolizumab isoform species The present invention provides a vedolizumab composition containing a basic isoform. In some embodiments, a basic isoform is provided. Compositions with reduced species levels are provided herein by method (e.g., Section III) This can be obtained by (see and examples). In some embodiments, basic a Compositions in which the level of isoform species is reduced are provided herein by methods (for example, (See Section III) is generated by. Therefore, in one aspect, this specification Provided in this document is a composition comprising vedolizumab, wherein the basic iso in this composition Allow sufficient time for the foam species level to decrease, then leave the bed at a pH above 6.5. A method comprising, in particular, incubating a composition containing zumab, which is produced , it is a composition. Following incubation, a composition containing vedolizumab is prepared, for example, It reduces the levels of process-derived impurities and / or product-derived impurities in the composition. It may optionally be subjected to further purification steps designed accordingly.

[0256] In another embodiment, what is provided herein includes vedolizumab or its antigen-binding moiety. A composition wherein, by the above method, for example, the antibody is subjected to low pH conditions during the purification process. This composition is obtained by limiting the period of exposure.

[0257] In some embodiments, provided herein are vedolizumab or its antigen A composition comprising a binding portion, wherein the composition comprises, in particular, (i) vedolizumab, or clarified cell culture obtained from cultures of recombinant host cells expressing the antigen-binding portion. (ii) to provide the harvested products of the nutrient, and (ii) vedolizumab or its antigen-binding portion Purification is performed from the harvested product of the cell culture, where the antibody or its antigen-binding portion pH 3.5 or lower (for example, pH 2.5 to 3.5, pH less than 3.0, or pH below 3.5) (Full) Within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 3 hours, within 5 hours, Within 7 hours, within 10 hours, or within 12 hours of exposure, this anti-α4β7 antibody or The antigen-binding region shows a reduced level of basic isoform species compared to the control. (Determined by CEX), this control is an anti-α4β7 antibody produced in the same manner. This is a composition containing the antigen-binding portion, and this antibody has a pH of 3.5 or lower (for example, pH 2.5 to 3.5, pH less than 3.0, or pH less than 3.5) for a long period of time, i.e., 2 Over 0 minutes, over 30 minutes, over 45 minutes, over 1 hour, over 3 hours, over 5 hours, over 7 hours, over 10 hours, Alternatively, the composition can be obtained by a method that includes exposure for more than 12 hours.

[0258] In some embodiments, provided herein are vedolizumab or its antigen A composition comprising a binding portion, wherein the composition comprises, in particular, (i) vedolizumab, or clarified cell culture obtained from cultures of recombinant host cells expressing the antigen-binding portion. (ii) to provide the harvested products of the nutrient, and (ii) vedolizumab or its antigen-binding portion Purification is performed from the harvested product of the cell culture, where the antibody or its antigen-binding portion pH 4.0 or lower (for example, pH 3.6-4.0), for 20 minutes, 30 minutes, or 45 minutes. Within, within 1 hour, within 3 hours, within 5 hours, within 10 hours, within 12 hours, over 15 hours Within 18 hours or 24 hours of exposure, this anti-α4β7 antibody or its antigenicity The combined portion shows a decrease in the level of basic isoform species compared to the control (CEX). (determined by this method), this control is an anti-α4β7 antibody or its antigen prepared by the same method. The composition contains a binding site, and this antibody is suitable for a pH of 4.0 or lower (for example, pH 3.6-4). 0) For long periods of time, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, more than 3 hours, 5 hours If exposed for more than 10 hours, more than 12 hours, more than 15 hours, more than 18 hours, or more than 24 hours This composition can be obtained by a method that includes [a certain action].

[0259] In another embodiment, provided herein are vedolizumab or its antigen-binding portion. A composition comprising, in particular, (i) vedolizumab or its anti- Harvesting of clarified cell cultures obtained from cultures of recombinant host cells expressing the primordial region. (ii) providing the substance, and (ii) vedolizumab or its antigen-binding portion, in the cell culture This involves purification from the harvested material, where the antibody or its antigen-binding portion is 4.5 or less. To the pH (for example, pH 4.1-4.5), within 3 hours, within 5 hours, within 10 hours, 1 Within 2 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours or within 96 hours of exposure, this anti-α4β7 antibody or its antigen-binding portion is controlled In comparison, the level of basic isoform species is lower (determined by CEX). (The control includes an anti-α4β7 antibody or its antigen-binding moiety prepared by the same method.) This composition contains an antibody that remains in a pH of 4.5 or lower (for example, pH 4.1-4.5) for an extended period of time. That is, more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours, including It is a composition obtainable by law.

[0260] In another embodiment, provided herein are vedolizumab or its antigen-binding portion. A composition comprising, in particular, (i) vedolizumab or its anti- Harvesting of clarified cell cultures obtained from cultures of recombinant host cells expressing the primordial region. (ii) providing the substance, and (ii) vedolizumab or its antigen-binding portion, in the cell culture This involves purification from the harvested material, where the antibody or its antigen-binding portion is 5.0 or less. To the pH (for example, pH 4.6-5.0), within 3 hours, within 5 hours, within 10 hours, 1 Within 2 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours or within 96 hours of exposure, this anti-α4β7 antibody or its antigen-binding portion is controlled In comparison, the level of basic isoform species is lower (determined by CEX). (The control includes an anti-α4β7 antibody or its antigen-binding moiety prepared by the same method.) This composition contains an antibody that, when exposed to a pH of 5.0 or lower (for example, pH 4.6-5.0) for an extended period of time, That is, more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours, including It is a composition obtainable by law.

[0261] In another embodiment, provided herein are vedolizumab or its antigen-binding portion. A composition comprising, in particular, (i) vedolizumab or its anti- Harvesting of clarified cell cultures obtained from cultures of recombinant host cells expressing the primordial region. (ii) providing the substance, and (ii) vedolizumab or its antigen-binding portion, in the cell culture This involves purification from the harvested material, where the antibody or its antigen-binding portion is 5.5 or less. To the pH (for example, pH 5.1-5.5), leave for 3 hours, 5 hours, 10 hours, 1 Within 2 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours or within 96 hours of exposure, this anti-α4β7 antibody or its antigen-binding portion is controlled In comparison, the level of basic isoform species is lower (determined by CEX). (The control includes an anti-α4β7 antibody or its antigen-binding moiety prepared by the same method.) This composition contains an antibody that remains in a pH of 5.5 or lower (for example, pH 5.1-5.5) for an extended period of time. That is, more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours, including It is a composition obtainable by law.

[0262] In some embodiments of the aforementioned model, the antibody or its antigen-binding portion is in SEQ ID NO: 1 A heavy chain variable region containing the amino acid sequence described, and a light chain containing the amino acid sequence described in SEQ ID NO: 5. Includes a variable chain region.

[0263] In some embodiments of the aforementioned model, an anti-α4β7 antibody, or its antigen-binding portion, is used to septically evaluate the antibody. The manufacturing steps are protein A chromatography and anion exchange chromatography. cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography This includes romatography and one or more combinations thereof.

[0264] In some embodiments of the aforementioned model, an anti-α4β7 antibody or its antigen-binding moiety is expressed. The host cells used are GS-CHO cells. In other embodiments, the host cells are DHFR- These are CHO cells.

[0265] In some embodiments of the aforementioned aspects, the composition is less than 16%, less than 15%, and 14%. Basicity of less than %, less than 13%, less than 12%, less than 11%, less than 10%, or less than 9% Includes isoform species.

[0266] In some embodiments of the aforementioned aspects, the composition is less than 4%, less than 3%, and less than 2%. or containing less than 1% of the basic isoform peak 2.

[0267] In some embodiments, the basic species of vedolizumab provided herein is composed A vedolizumab composition containing 15% or less of vedolizumab isoforms in the substance. For example. However, in some embodiments, provided herein, the percentages are approximately 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% The following are basic isoforms at levels of 4% or less, 3% or less, 2% or less, or 1% or less. This is a vedolizumab composition containing seeds.

[0268] In some embodiments, the level of basic isoform species in the vedolizumab composition is , about 1%~15%, 1%~14%, 1%~13%, 1%~12%, 1%~11%, 1%~ 10%, 1%~9%, 1%~8%, 1%~7%, 1%~6%, 1%~5%, 1%~4%, The amount is 1% to 3%, or 1% to 2%. In other embodiments, the base in the vedolizumab composition The levels of sex isoform species are approximately 2%-11%, 3%-11%, 4%-11%, and 5%- 11%, 6%~11%, 7%~11%, 8%~11%, 9%~11%, or 10%~1 It is 1%. In other embodiments, the level of basic isoform species in the vedolizumab composition The percentages are approximately 1%~10%, 2%~10%, 3%~10%, 4%~10%, 5%~10%, and 6%. These are %~10%, 7%~10%, 8%~10%, or 9%~10%. In other embodiments... The level of basic isoform species in the vedolizumab composition is approximately 1% to 9%, 2% to 9%, 3%~9%, 4%~9%, 5%~9%, 6%~9%, 7%~9%, or 8%~9% This is %. In other embodiments, the level of basic isoform species in the vedolizumab composition. These range from approximately 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, and so on. In other embodiments, the basic isophore in the vedolizumab composition is 7% to 8%. The levels of the species are approximately 1%~7%, 2%~7%, 3%~7%, 4%~7%, 5%~7%, and In other embodiments, the basic isophore in the vedolizumab composition is 6% to 7%. The levels of the species are approximately 1%~6%, 2%~6%, 3%~6%, 4%~6%, or 5%~6%. This is %. In other embodiments, the level of basic isoform species in the vedolizumab composition. These are approximately 1%-5%, 2%-5%, 3%-5%, or 4%-5%. (Example implementation) In this state, the level of basic isoform species in the vedolizumab composition is approximately 5% or less. .

[0269] In some embodiments, the percentage of basic isoform species in the vedolizumab composition The stage is incubated at a pH above 6.5 as described herein. Furthermore, the basic isoform species in the vedolizumab composition produced by the same method - Approximately 1% or more per cent, for example, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 6% or more, 7% or more, 8 % or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 1 5% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% It will decrease by more than 100%.

[0270] The percentage of basic isoform species in a composition containing vedolizumab is CEX- It can be determined by any suitable method, including but not limited to HPLC. In this embodiment, the anti-α4β7 antibody or its antigen-binding moiety is provided herein. For example, a low basic species composition containing vedolizumab, and this composition contains less than 16%, 1 Less than 5%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, or 9% This includes an anti-α4β7 antibody of less than 100%, or the total basic isoform species of its antigen-binding moiety. The basic isoform species of the anti-α4β7 antibody or its antigen-binding moiety is the major isoform. As determined by CEX, this anti-α4β7 antibody has a net positive charge relative to the foam. Alternatively, the antigen-binding portion includes a heavy chain variable region containing SEQ ID NO: 1, and a light chain containing SEQ ID NO: 5. It includes a variable region. In some embodiments, this composition is a first basic isoform. It includes peak (BP1) and a second basic isoform peak (BP2). In such embodiments, this composition contains less than 2% BP2, less than 1.5% BP2, and 1 Includes less than % BP2, or less than 0.7% BP2. In some embodiments, this set The finished product contains BP2 at 1.5%~2.5%, 1.2%~2.2%, 1%~2%, and 1% BP2 of ~1.8%, BP2 of 1%~1.6%, BP2 of 1%~1.5%, BP2 of 0.8%~1 BP2 at 0.8%, BP2 at 0.8% to 1.6%, BP2 at 0.8% to 1.4%, 0.8% BP2 of ~1.2%, BP2 of 0.8%~1%, BP2 of 0.7%~1.7%, 0.7% BP2 of ~1.5%, BP2 of 0.7%~1.3%, BP2 of 0.7%~1%, BP2 of 0.6% BP2 of ~1.6%, BP2 of 0.6%~1.4%, BP2 of 0.6%~1.2%, 0. BP2 of 6%~1%, BP2 of 0.6%~0.8%, BP2 of 0.5%~1.5%, 0. BP2 of 5% to 1.3%, BP2 of 0.5% to 1%, or BP2 of 0.5% to 0.8% Includes.

[0271] In some embodiments, the ratio of BP1 to BP2 in the composition is at least 3 (for example, At least 5, at least 6, at least 7, at least 8, at least 9, or fewer At most 10). In some embodiments, the ratio of BP1 to BP2 is 2 to 12, 2 to 10, 2 vs 8, 2 vs 6, 2 vs 4, 3 vs 12, 3 vs 11, 3 vs 9, 3 vs 6, 3 vs 5, 3 vs 4 4 vs 12, 4 vs 11, 4 vs 10, 4 vs 8, 4 vs 6, 4 vs 5, 5 vs 12, 5 vs 11, 5 vs 10, 5 vs 9, 5 vs 8, 5 vs 6, 6 vs 12, 6 vs 11, 6 vs 10, 6 vs 8, 6 vs 7, 7 vs 12, 7 vs 11, 7 vs 10, 7 vs 9, 7 vs 8, 8 vs 12, 8 vs 11, 8 vs 10, 8 vs 9, The scores are 9 to 12, 9 to 11, or 9 to 10.

[0272] In some embodiments, the composition of low basic species is less than 8% (e.g., less than 7%, 6%) The total basic isoform species of anti-α4β7 antibodies (e.g., less than 5%) Contains dolizumab. In some embodiments, the low basic species composition is 4% to 8%, 4% ~7.5%, 4%~7%, 4%~6.5%, 4%~6%, 4%~5.5%, 4%~5%, 5%~8%, 5%~7.5%, 5%~7%, 5%~6.5%, 5%~6%, 6%~8%, Total basic isoform species of anti-α4β7 antibodies: 6% to 7.5%, or 6% to 7% ( For example, it includes vedolizumab.

[0273] In some embodiments, the aforementioned composition is an anti-α4β7 antibody, or its antigen-binding moiety. A pharmaceutical composition comprising (e.g., vedolizumab) and a pharmaceutically acceptable carrier or excipient. It can be incorporated into an object. Therefore, in some embodiments, what is provided herein is , a low-basic species of anti-α4β7 antibody, or its antigen-binding moiety (e.g., vedolizumab), and It is a pharmaceutical composition containing a pharmaceutically acceptable carrier. This antibody preparation remains in liquid form. It may be a lyophilized or freeze-dried antibody preparation. In one embodiment, the dried The freeze-dried antibody preparations are available in 150mg, 180mg, 240mg, 300mg, and 360mg doses. It is supplied in a single-dose vial containing 450 mg or 600 mg of anti-α4β7 antibody, and It may be reconstituted with a liquid such as sterile water. In another embodiment, an anti-α4β7 antibody, for example. Vedolizumab, for example, will be administered to the person who needs it until it is given to them in a container, Stable liquid pharmaceuticals stored at approximately 2-8°C in vials, syringes, or cartridges. It is a composition. In some embodiments, the syringe or cartridge contains 54 mg. A single dose of antibody may be provided in the form of 108 mg, 160 mg, or 216 mg. In the embodiment, a recomposed lyophilized preparation of anti-α4β7 antibody or a stable liquid pharmaceutical composition The substances are not limited to amino acids (for example, arginine, histidine, and / or (e.g., histidine monohydrochloride), sugar (e.g., sucrose), surfactant (e.g., polysorbate) One or more of the following: β-80), and / or buffer (e.g., citrate, phosphate, etc.) The above excipients may be included. In one embodiment, a reconfigured lyophilized anti-α4β7 antibody is used. The preparation or stable liquid pharmaceutical composition contains L-arginine, L-histidine, and L-histidine. The present invention comprises monohydrochloride, sucrose, and / or polysorbate 80. In another embodiment, the anti A recomposed lyophilized preparation or stable liquid pharmaceutical composition of α4β7 antibody is citrate. , containing arginine, histidine, and / or polysorbate 80. Anti-α4β7 antibody Additional formulations and uses are, for example, U.S. Patent No. 9,764,033 and U.S. Patent No. 10. It is described in Patent No. 040,855. The entirety of the contents of each of the aforementioned patents is referenced herein. It is incorporated by.

[0274] V. Preparation of vedolizumab compositions containing low levels of host cell proteins In some embodiments, the present invention relates to a bedridy having a reduced amount of host cell protein. A method for producing a composition containing zumab is provided. This method is compared to standard operating conditions. Using AEX buffer with reduced conductivity (e.g., loading buffer) can disrupt standard operations. Compared to vedolizumab compositions produced under the specified conditions, the level of host cell proteins (HCPs) was significantly higher. This is based on the remarkable discovery that a vedolizumab composition with reduced performance can be obtained.

[0275] Therefore, in one embodiment, the present invention relates to a condition in which the amount of host cell protein (HCP) is reduced. This invention provides a method for producing a composition containing dolizumab, which includes vedolizumab and HCP. The method involves contacting a sample containing with an anion exchange resin in the presence of a loading buffer. Here, the conductivity of this loading buffer is lower compared to standard buffer conditions. This involves collecting flow-through material from anion exchange resin, and The flow-through includes vedolizumab and a reduced amount of HCP. Exemplary example: In the application method, AEX does not bind vedolizumab to the AEX resin, and there is no separate dissolution step. It runs in flow-through mode, and the collected material is then processed into a flow-through material.

[0276] Standard operating range of buffer conductivity for anion exchange (AEX) (e.g., anion exchange Q membrane absorption) The radiation level (through the attachment device) is approximately 11-15 mS / cm (average approximately 13.6 mS / cm). Therefore, in some embodiments, the standard AEX buffering conditions are approximately 11 mS / cm or higher. , approximately 12 mS / cm or higher, approximately 13 mS / cm or higher, approximately 14 mS / cm or higher, or approximately 15 The loading buffer includes a conductivity of mS / cm or more. In some embodiments, The conductivity of a standard buffer solution is approximately 11 mS / cm to approximately 12 mS / cm, approximately 11 mS / cm to approximately 13 mS / cm, approximately 11 mS / cm to approximately 14 mS / cm, or approximately 11 mS / cm to approximately 1 It is 5 mS / cm. In some embodiments, the conductivity of a typical buffer is approximately 14 mS / cm. cm ~ approx. 15mS / cm, approx. 13mS / cm ~ approx. 15mS / cm, approx. 12mS / cm ~ approx. It is 15 mS / cm, or approximately 11 mS / cm to approximately 15 mS / cm. Several implementations In this state, the conductivity of standard buffer solutions is approximately 11 mS / cm, approximately 12 mS / cm, and approximately 13 mS / cm. This corresponds to a density of approximately 14 mS / cm or 15 mS / cm.

[0277] A deconductive AEX buffer used in the method described herein (e.g., AEX ro The lead buffer has lower conductivity compared to standard AEX buffer conditions. For example, In a particular embodiment, the loading buffer with reduced conductivity is approximately 15 mS / cm or less. Bottom, approx. 14mS / cm or less, approx. 13mS / cm or less, approx. 12mS / cm or less, approx. 11mS / cm or less, approximately 10mS / cm or less, approximately 9mS / cm or less, approximately 8mS / cm or less, approximately 7m S / cm or less, approximately 6mS / cm or less, approximately 5mS / cm or less, approximately 4mS / cm or less, approximately 3m It has an electrical conductivity of S / cm or less, or about 2 mS / cm or less. In some embodiments, Loading buffer with reduced conductivity has a conductivity of approximately 11 mS / cm or less.

[0278] In certain embodiments, the load buffer with reduced conductivity is approximately 1 mS / cm to approximately 1 1mS / cm, approx. 2mS / cm ~ approx. 11mS / cm, approx. 3mS / cm ~ approx. 11mS / cm , approx. 4mS / cm ~ approx. 11mS / cm, approx. 5mS / cm ~ approx. 11mS / cm, approx. 6mS / cm ~ approx. 11mS / cm, approx. 7mS / cm ~ approx. 11mS / cm, approx. 8mS / cm ~ approx. 11 mS / cm, approximately 9 mS / cm to approximately 11 mS / cm, or approximately 10 mS / cm to approximately 11 mS It has a conductivity of / cm (including the range within one or more of the above ranges). Several implementations Morphologically, loading buffer with reduced conductivity is approximately 11 mS / cm to approximately 12 mS / cm. m, approximately 11 mS / cm to approximately 13 mS / cm, approximately 11 mS / cm to approximately 14 mS / cm, also The range is approximately 11 mS / cm to approximately 14.5 mS / cm (including the range of one or more of the above ranges) It has a conductivity of the following: In certain embodiments, a loading buffer with reduced conductivity is approximately 1mS / cm ~ approx. 2mS / cm, approx. 1mS / cm ~ approx. 3mS / cm, approx. 1mS / cm ~ approx. 4mS / cm, approx. 1mS / cm ~ approx. 5mS / cm, approx. 1mS / cm ~ approx. 6mS / cm, approx. 1mS / cm~Approx. 7mS / cm, Approx. 1mS / cm~Approx. 8mS / cm, Approx. 1mS / cm~Approx. 9 mS / cm, approximately 1 mS / cm to approximately 10 mS / cm, or approximately 1 mS / cm to approximately 11 mS It has an conductivity of / cm (including a range within one or more of the above ranges).

[0279] In certain embodiments, the loading buffer with reduced conductivity is approximately 1 mS / cm, approximately 1 .5mS / cm, approx. 2mS / cm, approx. 2.5mS / cm, approx. 3mS / cm, approx. 3.5mS / cm, approx. 4mS / cm, approx. 4.5mS / cm, approx. 5mS / cm, approx. 5.5mS / cm, Approx. 6mS / cm, Approx. 6.5mS / cm, Approx. 7mS / cm, Approx. 7.5mS / cm, Approx. 8mS / cm, approx. 8.5mS / cm, 9mS / cm, 9.5mS / cm, 10mS / cm, 10 .5mS / cm, 11mS / cm, 11.5mS / cm, 12mS / cm, 12.5mS / cm, 13mS / cm, 13.5mS / cm, 14mS / cm, 14.5mS / cm, Alternatively, it has a conductivity of 15 mS / cm.

[0280] In some embodiments, this method involves the application of a loading buffer containing vedolizumab. This may further include applying a washing buffer to the AEX resin. Several implementations In this configuration, the elution buffer has the same conductivity as the loading buffer. In other embodiments, The washing buffer has increased conductivity compared to the loading buffer. In other embodiments The washing buffer has lower conductivity compared to the loading buffer.

[0281] In some embodiments, the loading buffer is sodium chloride and / or phosphoric acid. Contains sodium. In exemplary embodiments, the loading buffer is 40–70 mM Na Cl (for example, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM or 7 It contains 0 mM NaCl). In some embodiments, the loading buffer is 55-6 Contains 5 mM NaCl. Additionally, or alternatively, the loading buffer contains sodium phosphate. It may contain um. In an exemplary embodiment, the loading buffer is 20-50 mM Sodium phosphate (for example, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM) It contains mM or 50 mM sodium phosphate. In some embodiments, Rhodi The buffer solution contains 35-45 mM sodium phosphate. In some embodiments, The loading buffer should have a pH of 6.5 or higher, for example, pH 6.5-8.5, pH 7.0 These are typically around 7.5, with a pH of 6.8-7.4. In some embodiments, a loading buffer is used. The liquid is approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3. 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, The pH is 8.4 or 8.5.

[0282] In some embodiments, the following are provided herein: low conductivity in flow-through mode Contacting the AEX resin equilibrated in buffer with a solution containing antibodies, and flow A method for purifying α4β7 antibodies, such as vedolizumab, including collecting raw material. This is the method. In one embodiment, the low conductivity solution is made of NaCl and a buffer solution with a pH of 6.5 to 8.5. It contains a mixture of the following. In some embodiments, the low conductivity solution is 5-15 mS / cm, 5- It has an electrical conductivity of 11 mS / cm, 7-10 mS / cm, or approximately 10 mS / cm.

[0283] In some embodiments, the anion exchange resin is formatted as an anion exchange membrane. In some embodiments, the anion exchange resin is used in an anion exchange chromatograph. It is formatted as an anion exchange resin. In some embodiments, an anion exchange resin It contains a quaternary amine functional group. Examples of AEX resins include, but are not limited to, , Mustang Q(Pall Corporation,Port Washing ton, NY), Sartobind Q (Sartorius GmbH, Goett Examples of AEX resins include, for example, ingen (Germany). , Eshmuno Q resin(EMD Millipore, Burlingto n, MA) and Nuvia Q resin (Bio-Rad, Hercules, CA) ) are some examples.

[0284] HCP is a host cell used to produce vedolizumab or its antigen-binding moiety. It may originate from the following. For example, in some embodiments, vedolizumab is derived from the Chinese hamster. Produced in ovarian (CHO) cells, HCP is a CHO cell protein. In terms of application, HCP is used in CHO cells that lack dihydrofolate reductase (DHFR) expression. It originates from. In certain embodiments, HCP lacks glutamine synthetase (GS) expression. Derived from CHO cells.

[0285] In certain embodiments, the amount of HCP in the flow-through is approximately 8 ppm or less, or approximately 7.5 ppm. m or less, about 7ppm or less, about 6.5ppm or less, about 6ppm or less, about 5.5ppm or less, About 5ppm or less, about 4.5ppm or less, about 4ppm or less, about 3.5ppm or less, about 3pp m or less, approximately 2.5 ppm or less, approximately 2 ppm or less, approximately 1.5 ppm or less, or approximately 1 ppm The following applies:

[0286] In some embodiments, the amount of HCP in the flow-through is 1 ppm to 8 ppm, 2 ppm. pm~8ppm, 3ppm~8ppm, 4ppm~8ppm, 5ppm~8ppm, 6p This range is between 8 ppm or 7 ppm to 8 ppm (including one or more of the above ranges). In some embodiments, the amount of HCP in the flow-through is 1 ppm to 2 ppm, 1 ppm m~3ppm, 1ppm~4ppm, 1ppm~5ppm, 1ppm~6ppm, 1pp m to 7 ppm, or 1 ppm to 8 ppm (including one or more of the above ranges). In several embodiments, the amount of HCP in the flow-through is 1 ppm to 3 ppm, 3 ppm It is approximately 5 ppm, or 5 ppm to 7 ppm.

[0287] In certain embodiments, the amount of HCP in the flow-through is the standard conductivity (e.g., 11-1) Using a loading buffer with a conductivity of 5 mS / cm or higher, the same sample was used. When this method is performed, the amount of HCP in the flow-through material generated is small At least 0.5%, at least 1%, at least 2%, at least 5%, and less Approximately 10%, at least 15%, at least 20%, at least 25%, and less Approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, and less Approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, and less Approximately 70%, at least 75%, at least 80%, at least 85%, and less Both approximately 90%, at least approximately 95%, or at least approximately 98% or more than approximately 98%, low It has been reduced.

[0288] In certain embodiments, the amount of HCP in the flow-through is approximately 0.5% to approximately 50%, and approximately 1%. ~50%, 2%~50%, 5%~50%, 10%~50%, 15%~ 50%, approximately 20% to approximately 50%, approximately 25% to approximately 50%, approximately 30% to approximately 50%, approximately 35% to approximately Reduced by 50%, approximately 40% to approximately 50%, or approximately 45% to approximately 50%. In certain embodiments The amount of HCP in the flow-through is equal to the standard conductivity (e.g., approximately 11-15 mS / cm). When this method is performed using the same sample with the loading buffer, the following is generated. Approximately 50% to 55%, approximately 50% to 60%, and approximately 50% to approximately 65%, approximately 50% to approximately 70%, approximately 50% to approximately 75%, approximately 50% to approximately 80%, approximately 50% to approximately 85%, approximately 50% to approximately 90%, approximately 50% to approximately 95%, or approximately 50% to approximately 98% It will be reduced by %.

[0289] In certain embodiments, the amount of HCP in the flow-through is approximately 1% to approximately 10%, approximately 10% to Approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 40% to approximately 50%, approximately 50% to Approximately 60%, approximately 60% to approximately 70%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, or approximately 9 It is reduced by 0% to approximately 98%. In certain embodiments, the amount of HCP in the flow-through is standard Using a loading buffer with conductivity (e.g., approximately 11-15 mS / cm), the same experiment was performed. For the amount of HCP in the flow-through generated when this method is performed using the following materials Approximately 0.5% to 1%, approximately 1% to 2%, approximately 2% to 5%, approximately 5% to 10%, approximately 10% ~15%, 15%~20%, 20%~25%, 25%~30%, 30% ~35%, 35%~40%, 40%~45%, 45%~50%, 50% ~55%, 55%~60%, 60%~65%, 65%~70%, 70% ~75%, 75%~80%, 80%~85%, 85%~90%, 90% It will be reduced by approximately 95%, or approximately 95% to 98%.

[0290] Samples containing vedolizumab and HCP may optionally undergo, for example, affinity chromatography. Chromatography, cation exchange chromatography, hydrophobic interaction chromatography, Lamic hydroxyapatite (CHT) chromatography, and mixed-mode chromatography After one or more additional purification steps such as Graphy, or a combination thereof, the feeding movement It is derived from physical cell culture. Furthermore, it contains vedolizumab according to the AEX method described herein. The level of HCP in a sample can be measured, for example, by affinity chromatography or cation exchange chromatography. Conversion chromatography, hydrophobic interaction chromatography, ceramic hydroxya Tight (CHT) chromatography, and mixed-mode chromatography, or the The results are optionally further reduced by one or more additional purification steps, including a combination of these steps. obtain.

[0291] Therefore, the methods described herein, in some embodiments, include vedolizumab. Includes one or more additional purification steps to further reduce the level of HCP in the sample. However, this may also be the case. In one embodiment, the present invention uses the AEX method described herein, and further 1 A method to reduce the level of HCP in a vedolizumab-containing composition, including one or more additional purification steps. A method for causing this is provided. One or more additional purification steps are provided for the AEX method described herein. This can be performed before or after. In some embodiments, one or more additional purification steps This includes one or more chromatographic separations. In exemplary embodiments, one or more additional The purification process involves affinity chromatography (for example, protein A chromatography). Chromatography, cation exchange chromatography, hydrophobic interaction chromatography , ceramic hydroxyapatite (CHT) chromatography, or mixed mode chromatography Examples include rhombustography, or a combination of these.

[0292] In exemplary embodiments, the present invention reduces the level of HCP in a composition containing vedolizumab. This provides a method for causing a decrease in blood pressure, and this method provides a composition comprising vedolizumab and HCP. and affinity chromatography, mixed-mode chromatography, and / or Vedolizumab was purified from HCP by cation exchange chromatography. Furthermore, by implementing the AEX method described herein, HCPs are reduced to bedrids This includes further purification of the mab. In one embodiment, the loading of the AEX method described herein The material contains a cation exchange eluate. Using the method described above, 11-15 mS / c Using an anion exchange buffer with a conductivity of m or higher, in the composition resulting from the implementation of the same method Composition containing vedolizumab with reduced HCP levels compared to the existing HCP levels It can generate objects.

[0293] In another exemplary embodiment, the present invention relates to the HCP level in a composition comprising vedolizumab. The present invention provides a method for reducing the following: the method involves a composition comprising vedolizumab and HCP. To provide, affinity chromatography, cation exchange chromatography and / or hydroxyapatite chromatography (e.g., ceramic hydroxyapatite chromatography) By performing apatite (CHT) chromatography, bedrids are obtained from HCP. By purifying the mab and further performing the AEX method described herein, HCP This includes further purifying vedolizumab from the present invention. In one embodiment, A as described herein The load material for the EX method contains a hydroxyapatite chromatography eluate. Using the method described above, an anion exchange buffer with a conductivity of 11-15 mS / cm or higher is used. Then, compared with the level of HCP present in the composition resulting from the implementation of the same method, A composition containing vedolizumab with reduced levels of [the substance] can be produced.

[0294] The contents of vedolizumab and / or host cell protein content are determined by HCP ELISA. Chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MA) LLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoparticle tracking analysis N Nanoscale measurement by companies such as TA, NanoSight Ltd, Wiltshire, UK Measured by any method known in the art, including but not limited to (determinative) obtain.

[0295] In some embodiments, this method involves one or more pharmaceutically acceptable carriers or excipients. By a process including ultrafiltration and / or diafiltration into a buffer containing the agent This further involves processing AEX flow-through material and replacing the buffer.

[0296] VI. Vedolizumab composition containing reduced host cell proteins In some embodiments, the present invention relates to a bedridden cell containing reduced host cell proteins. The present invention provides a zumab composition. In some embodiments, the composition is reduced in host cell protein. The material is produced by the methods provided herein (see, for example, Section V). (and). Therefore, in one embodiment, a composition comprising vedolizumab is provided herein. Herein, this composition loads a sample containing vedolizumab and HCP slowly. This involves contact with the anion exchange resin in the presence of a buffer, where this loading buffer is located. The solution has lower conductivity compared to standard buffer conditions, and the anion exchange resin... The process involves collecting flow-through material, and this flow-through material is vedolizumab This is produced by the inclusion of a reduced amount of HCP, following the elution from the AEX resin. Then, the flow-through material is processed as desired, and one or more pharmaceutically acceptable elution buffers are used. Replace the buffer containing the carrier or excipient, thereby reducing the amount of HCP in the drug. A chemical composition can be formed. This buffer exchange step is, for example, ultrafiltration and / or diaphragm This can be carried out using standard methods, including filtering.

[0297] HCP is a host cell used to produce vedolizumab or its antigen-binding moiety. It may originate from the following. For example, in some embodiments, vedolizumab is derived from the Chinese hamster. Produced in ovarian (CHO) cells, HCP is a CHO cell protein. In terms of application, HCP is used in CHO cells that lack dihydrofolate reductase (DHFR) expression. It originates from. In certain embodiments, HCP lacks glutamine synthetase (GS) expression. Derived from CHO cells.

[0298] In certain embodiments, the amount of HCP in this composition is about 8 ppm or less, and about 7.5 ppm. Below, about 7ppm or less, about 6.5ppm or less, about 6ppm or less, about 5.5ppm or less, about 5ppm or less, approximately 4.5ppm or less, approximately 4ppm or less, approximately 3.5ppm or less, approximately 3ppm less than about 2.5ppm, less than about 2ppm, less than about 1.5ppm, or less than about 1ppm It is below.

[0299] In some embodiments, the amount of HCP in this composition is 1 ppm to 8 ppm, 2 ppm m~8ppm, 3ppm~8ppm, 4ppm~8ppm, 5ppm~8ppm, 6pp m to 8 ppm, or 7 ppm to 8 ppm (including one or more of the above ranges). In some embodiments, the amount of HCP in this composition is 1 ppm to 2 ppm, 1 ppm to 3ppm, 1ppm~4ppm, 1ppm~5ppm, 1ppm~6ppm, 1ppm~ 7 ppm, or 1 ppm to 8 ppm (including one or more of the above ranges). How many? In that embodiment, the amount of HCP in this composition is 1 ppm to 3 ppm, 3 ppm to 5 ppm. This is pm, or 5 ppm to 7 ppm.

[0300] In certain embodiments, the amount of HCP in this composition is such that the standard conductivity is (e.g., 11-15) The same sample was used with a loading buffer having a conductivity of mS / cm or higher. The amount of HCP in the composition produced by the method is at least about 0.5%, At least about 1%, at least about 2%, at least about 5%, at least about 10%, at least Approximately 15%, at least 20%, at least 25%, at least 30%, and less Approximately 35%, at least 40%, at least 45%, at least 50%, and less Both approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, less Approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, and less Both have been reduced by approximately 95%, or at least approximately 98%, or more than approximately 98%.

[0301] In certain embodiments, the amount of HCP in this composition is about 0.5% to about 50%, about 1% to Approximately 50%, approximately 2% to approximately 50%, approximately 5% to approximately 50%, approximately 10% to approximately 50%, approximately 15% to approximately 5 0%, approximately 20% to approximately 50%, approximately 25% to approximately 50%, approximately 30% to approximately 50%, approximately 35% to approximately 5 The reduction is 0%, approximately 40% to approximately 50%, or approximately 45% to approximately 50%. In certain embodiments, The amount of HCP in the flow-through material is the standard conductivity (e.g., approximately 11-15 mS / ). The same sample was used with a loading buffer having a conductivity of 1 cm or more. Approximately 50% to 55% of the amount of HCP in the composition produced by the method, approximately 50% to 55% Approximately 60%, approximately 50% to approximately 65%, approximately 50% to approximately 70%, approximately 50% to approximately 75%, approximately 50% to Approximately 80%, approximately 50% to approximately 85%, approximately 50% to approximately 90%, approximately 50% to approximately 95%, or approximately 5 It is reduced by 0% to approximately 98%.

[0302] In certain embodiments, the amount of HCP in the composition is about 1% to about 10%, about 10% to about 20% %, approximately 20% to 30%, approximately 30% to 40%, approximately 40% to 50%, approximately 50% to 60% %, approximately 60% to 70%, approximately 70% to 80%, approximately 80% to 90%, or approximately 90% It is reduced by approximately 98%. In certain embodiments, the amount of HCP in the flow-through material is Loading buffer with standard conductivity (e.g., conductivity of approximately 11-15 mS / cm or higher) The amount of HCP in the composition produced by the method performed using the same sample with a liquid. In contrast, approximately 0.5% to 1%, approximately 1% to 2%, approximately 2% to 5%, and approximately 5% to 10%. Approximately 10% to 15%, approximately 15% to 20%, approximately 20% to 25%, approximately 25% to 30%, Approximately 30% to 35%, approximately 35% to 40%, approximately 40% to 45%, approximately 45% to 50%, Approximately 50% to 55%, approximately 55% to 60%, approximately 60% to 65%, approximately 65% ​​to 70%, Approximately 70% to 75%, approximately 75% to 80%, approximately 80% to 85%, approximately 85% to 90%, It will be reduced by approximately 90% to 95%, or approximately 95% to 98%.

[0303] The vedolizumab content and / or host cell protein content were determined by HCP ELISA. Chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MA) LLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoparticle tracking analysis N Nanoscale measurement by companies such as TA, NanoSight Ltd, Wiltshire, UK Measured by any method known in the art, including but not limited to (determinative) obtain).

[0304] VII. Preparation of vedolizumab using mixed-mode chromatography This specification describes the process after elution from mixed-mode chromatography resins. This buffer maximizes the yield of dolizumab. Protein loss is due to high concentrations of rhodin. Conditions (for example, at least 14g / L, 15g / L, 17g / L, 10g / L, 25g) (At loading concentrations below / L, 30g / L, 35g / L or higher), for example, ceramic hydr This can occur during the washing step of mixed-mode purification using roxyapatite resin. To increase the loading capacity of a resin, such as ceramic hydroxyapatite resin. To improve the yield obtained as a result of vedolizumab, equilibration, loading, and The washing buffer can be optimized to reduce antibody loss when washing the column. Rather, the method described herein allows for a higher loading concentration into the mixed mode resin. This makes it possible to change the quality of the product in the final eluate (e.g., aggregate percentage). Furthermore, it increases the yield by 2-3% compared to standard mixed-mode buffers.

[0305] Therefore, in one embodiment, the present invention is used after elution from mixed-mode chromatography resin. A method for increasing the yield of vedolizumab, comprising mixed-mode chromatography. Equilibration of the resin with equilibration buffer, and solution containing vedolizumab and loading buffer. The mixture is loaded onto a mixed-mode chromatography resin, and vedolizumab is processed in mixed-mode chromatography. To bind to the chromatographic resin, mixed-mode chromatography with washing buffer. - Wash the resin and elute it with elution buffer in mixed mode chromatography to remove the vesicles from the resin. This includes eluting zumab, wherein this equilibration buffer, loading buffer and / or This provides a method in which the pH of the washing buffer is 7.0 or less. Mixed-mode chromatography - The standard buffer for this purpose can be made to a higher pH, i.e., pH 7.2 or higher. In some embodiments, an equilibration buffer having a pH of 7.0 or less is used. In the application method, a loading buffer with a pH of 7.0 or lower is used. Several applications are performed. In this configuration, a washing buffer with a pH of 7.0 or lower is used.

[0306] To prevent vedolizumab loss during the washing step of mixed-mode purification, 7.0 (unclear) A buffer pH within the full range may be used. For example, in some embodiments, equilibration buffer The pH of the solution, loading buffer, and / or washing buffer should be less than 7.0 and less than 6.9. Less than 6.8, less than 6.7, less than 6.6, less than 6.5, less than 6.4, less than 6.3, less than 6.2 Full, less than 6.1, less than 6.0, less than 5.9, less than 5.8, less than 5.7, less than 5.6, and It can be less than 5.5. For example, buffer solutions are approximately 7.0, 6.9, 6.8, 6.7, 6. 6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5. It may have a pH of 6 or 5.5. In some embodiments, the buffer solution is about 7.0 to 5 It has a pH in the range of 0.5. In other embodiments, the buffer solution has a pH in the range of about 7.0 to 6.0. It contains H. In other embodiments, the buffer solution has a pH in the range of about 7.0 to 6.5. In one embodiment, the buffer solution has a pH in the range of approximately 6.8 to 5.8. In another embodiment, In other embodiments, the buffer solution has a pH in the range of approximately 6.8 to 6.0. The pH is in the range of 6.8 to 6.5. In other embodiments, the buffer is approximately 6.6 to 6.8. It has a pH of [value].

[0307] Furthermore, or alternatively, mixed-mode chromatography for the purification of vedolizumab. The buffer used for equilibrating, loading, and / or washing the resin must have a total salt concentration of less than 70 mM. It can have degrees. For example, the buffer solution can have concentrations of less than 70 mM, less than 65 mM, less than 60 mM, and 55 mM. Less than M, less than 50mM, less than 45mM, less than 40mM, less than 35mM, or less than 30mM It may have a high salt concentration. In some embodiments, the buffer solution is about 70 mM, about 65 mM. M, approximately 60mM, approximately 55mM, approximately 50mM, approximately 45mM, approximately 40mM, approximately 35mM, also In one embodiment, the salt concentration is approximately 30 mM. In another embodiment, the buffer solution is in the range of 30 to 70 mM. It has a salt concentration. In some embodiments, the buffer has a salt concentration in the range of 40 to 65 mM. It has. In some embodiments, the buffer solution has a salt concentration in the range of 40 to 65 mM. In some embodiments, the buffer solution has a salt concentration in the range of 50–60 mM. In this embodiment, the buffer solution has a salt concentration in the range of 40-50 mM. In this configuration, the buffer solution has a salt concentration in the range of 45-55 mM. In some embodiments, Equilibration, loading, and CHT chromatography resin for vedolizumab purification. The salts present in the buffer used for washing and / or cleaning are sodium chloride and / or phosphoric acid. Contains sodium.

[0308] In some embodiments, mixed-mode chromatography is used for the purification of vedolizumab. The buffer used for equilibrating, loading, and / or washing the resin is less than 70 mM. The concentration of sodium chloride may be less than 70 mM, or less than 65 mM. Full, less than 60mM, less than 55mM, less than 50mM, less than 45mM, less than 40mM, 35m The sodium chloride concentration may be less than M or less than 30 mM. Several implementations In terms of morphology, this buffer solution is available in concentrations of approximately 70 mM, 65 mM, 60 mM, 55 mM, and 50 mM. Sodium chloride concentrations of M, approximately 45mM, approximately 40mM, approximately 35mM, or approximately 30mM. In other embodiments, this buffer solution contains a sodium chloride concentration in the range of 30 to 70 mM. It has a degree. In some embodiments, this buffer contains sodium chloride in the range of 40-65 mM. It has a lium concentration. In some embodiments, this buffer is in the range of 40 to 65 mM. It has a sodium chloride concentration. In some embodiments, this buffer is 50-60 mM. It has a sodium chloride concentration in the range of 40~ It has a sodium chloride concentration in the range of 50 mM. In some embodiments, this buffer is , having a sodium chloride concentration in the range of 45-55 mM. In some embodiments, as described above. The sodium chloride buffer may further contain sodium phosphate. Several implementations In this state, the aforementioned sodium chloride buffer contains 1-30 mM sodium phosphate, for example, 5 It also contains ~20 mM sodium phosphate, 10~20 mM sodium phosphate, etc. It is also acceptable. In an exemplary embodiment, the sodium chloride buffer is further divided into 1 mM, 2 mM, and 3 mM. mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20m It may also contain sodium phosphate at concentrations of M, 25 mM, or 30 mM.

[0309] The above buffer is used for equilibrating mixed-mode chromatography resins during vedolizumab purification. , can be used for loading and / or washing. In an exemplary embodiment, a mixed mode tree The lipid may be a ceramic hydroxyapatite resin.

[0310] In some embodiments, two of the equilibration, loading, and wash buffers are the same It has the same pH. In some embodiments, equilibration, loading, and washing buffer are used. The two have the same salt concentration. In some embodiments, equilibration, loading, and Two of the washing buffers have the same pH and the same salt concentration. In some embodiments, The equilibration, loading, and washing buffers all have the same pH. In this application, the equilibration, loading, and washing buffers all have the same salt concentration. In some embodiments, the equilibration, loading, and washing buffers all have the same pH and They have the same salt concentration.

[0311] In some embodiments, the mixed-mode chromatography resin is loaded and then recovered. Methods to increase the yield of vedolizumab include setting the column pH below 7 and pH 5.5-6.9. Alternatively, pH 6.5-6.8 and salts, for example, at concentrations of 40-60 mM or 45-55 mM. This method includes elution with NaCl. This method is suitable for pH levels below 7, pH 5.5-6.9, and This may further include washing the column with pH 6.5–6.8.

[0312] The buffers and methods described herein include equilibration, loading, and / or washing steps. The buffer solution has a pH above 7.0, and / or a salt concentration above 70 mM, and / or For the yield when performed using sodium chloride concentrations above 70 mM, the mixed This may improve the yield of vedolizumab eluted from mode chromatography columns. In several embodiments, the yield is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%. Improvements of 8%, 9%, 10%, 12%, 15%, 18%, and 20% or more are observed. Several implementations In terms of morphology, the yield improved by more than 2% compared to buffers with a pH greater than 7.0. In some embodiments, the yield is compared to a buffer having a pH greater than 7.0. The yield is improved by more than 3%. In some embodiments, the yield is obtained when the pH is greater than 7.0. The yield is improved by more than 4% compared to the buffer solution. In some embodiments, the yield is 7 Compared to buffers with a pH greater than 0, this shows an improvement of over 5%.

[0313] VIII. Method for evaluating the purity of a composition containing an anti-α4β7 antibody The purity of an antibody, for example, a composition containing vedolizumab, includes the methods described herein, but It can be evaluated by any appropriate method, not limited to the above.

[0314] (a) Evaluation of basic isoform species The present invention relates to a basic isoform in a composition containing vedolizumab or its antigen-binding moiety. Methods for adjusting the level of the main component in a composition containing vedolizumab, for example, by reducing it. Provides a method to adjust the level of essential isoforms, for example, by increasing them. The relative amounts of basic vedolizumab isoform species present in the mab composition, and the major vedolizumab The relative amount of lizumab isoform is described in detail in the Examples section, as positive It can be measured using on-exchange chromatography (CEX). The CEX method is a global The antibody species are fractionated according to their surface charge. After dilution to low ionic strength using the mobile phase, the test is performed. Place the sample in a CEX column, for example, with a suitable buffer, for example, 10 mM sodium phosphate, pH Dionex Pro-Pac™ WCX-10 column, equilibrated at 6.6 (The RMO Fisher Scientific, Waltham, MA (USA), etc. It may be injected into the solution. The antibody can be eluted using a sodium chloride gradient in the same buffer solution. Protein elution can be monitored at 280 nm, with peaks indicating acidity, basicity, or It is assigned to the category of major isoforms. The acidic peak is the major isoform. Elution from the column occurs with a shorter retention time than the peak, and the basic peak is the major isophore. Elutes from the column with a longer retention time than the peak. Proportion of major isoforms, acid The total proportion of sex species and the total proportion of basic species are reported. The main isophores of the sample are also reported. The retention time is compared to the retention time of the reference standard to determine compliance. In this case, the CEX-HPLC method is used. For example, vedolizumab and its acidity and / or The composition containing basic species was prepared using cation exchange chromatography as described above. Subsequently, the eluted peaks can be analyzed and separated using HPLC. In one embodiment, HPLC is the Agilent 1200 HPLC system (Agilent, San This may be performed using ta Clara (CA). Quantification is performed relative to the detected peak. Based on area percentage. CEX-HPLC profile of an example vedolizumab preparation. The file is shown in Figure 1.

[0315] In one embodiment, the CEX assay method involves diluting the test sample to a low ionic strength, 10 Inject mM sodium phosphate into a CEX column equilibrated at pH 6.6. The column is eluted using an NaCl gradient in the buffer, and the peak is monitored at 280 nm. This includes assigning the peaks as acidic, primary, or basic, and here The acidic peak elutes first with the shortest retention time, the main peak elutes second, and the basic peak elutes first. The compound elutes with the longest retention time, the peak area is quantified, and its amount is the sum of all peak areas. It will be calculated as a percentage.

[0316] (b) Assessment of host cell protein levels This invention relates to residual host cell proteins in a composition containing vedolizumab or its antigen-binding moiety. The present invention provides a method for adjusting the quality level, for example, by reducing it. In some embodiments, The amount of host cell protein present in the vedolizumab composition was determined using standard techniques. It can be measured using enzyme-linked immunosorbent assay (ELISA). Cygnus Te CHO HCP EL from chnologies(Southport,NC(USA)) Many ELISA kits designed for this purpose, such as the ISA Kit 3G, are commercially available. The host cell proteins in the test sample are immobilized polyclonal anti-CHOH. The captured protein can then be detected using a CP antibody. It can then be detected using a horseradish peroxidase-labeled version of the same antibody. In an exemplary embodiment, the captured peroxider is directly proportional to the concentration of CHO HCP. The amount of ze is the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB The HCP concentration can be measured colorimetrically at 450 nm using the following method. It can be determined by comparing it with the standard CHO HCP curve of the antibody preparation, etc. It is reported as a percentage of the total level of protein within. In another embodiment, HC P is a Rabbit-Rabbit (RaR) as exemplified herein. a) can be determined using the method. Polyclonal anti-CHOHCP antibodies are vedolizumab It is produced by immunizing rabbits with harvested material produced in null cells under similar manufacturing conditions. This antibody was then affinity-purified. Host CHO cell protein in vedolizumab sample The substance was captured using immobilized polyclonal anti-CHO HCP antibody, and then the same anti Biotin-labeled version of the body, followed by horseradish peroxidase-conjugated streptavid Detected using [a specific method]. Captured peroxids are directly proportional to the concentration of CHO HCP. The amount of ze is the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB The HCP concentration can be measured colorimetrically at 450 nm using the following method. Determined by comparing with the standard CHO HCP curve, the percentage of total protein It is reported as follows. In another embodiment, the HCP is a rabbit-goat as illustrated herein ( This can be determined using the Rabbit-Goat ("RaGo") method. Polyclonal The anti-CHO HCP antibody was manufactured in null cells using a manufacturing process similar to that of vedolizumab. The antibodies were produced by immunizing rabbits and goats with the manufactured harvest substance. The antibody pool was independently analyzed. Affinity purification was performed. The host cell proteins of the MLN0002 test sample were fixed. The polyclonal rabbit was captured using anti-CHO HCP antibodies, and then goat anti-CH O affinity purified antibody and donkey anti-goat IgG reagent (marked with horseradish peroxidase) It is detected by sequentially adding the identified substances. It is directly proportional to the concentration of CHO HCP. The amount of captured peroxidase is equal to the peroxidase substrate 3,3',5,5'-tetra It is measured colorimetrically at 450 nm using methylbenzidine (TMB). HCP The concentration is determined by comparing it to the CHO HCP standard curve included in the test kit, and the total vegetative state The concentration of lizumab is reported as (ng / mg).

[0317] CHO HCP suitable for use in relation to the various embodiments provided herein Ssey uses polyclonal anti-CHO HCP antibodies to capture HCP. It contains the peroxidase substrate 3,3',5,5'-tetramethylbenzidine ( Polyclonal antimicrobial agents (TMB) are converted into substances that can be quantified by colorimetric analysis at 450 nm. Detected after binding to a horseradish peroxidase-labeled version of the CHO HCP antibody. In one embodiment, it is suitable for use in relation to the various embodiments provided herein. The HCP ELISA used was an immobilized polyclonal anti-CHO HCP antibody, preferably C CH of ygnus Technologies (Southport, NC (USA)) O. Use the provided antibody to capture HCP in the HCP ELISA Kit 3G. This is the LISA method, which captures the protein, and the same antibody as horseradish peroxide. The amount of peroxidase detected and captured by the xidase-labeled version was 3, 3',5,5'-tetramethylbenzidine (TMB) is used for colorimetric analysis at 450 nm. The HCP concentration is then measured and subsequently determined by comparison with the CHO HCP standard curve.

[0318] (c) Evaluation of size variants In certain embodiments, chromatography produced using the techniques described herein. The levels of aggregates, monomers, and fragments in the sample are analyzed. In various embodiments, size exclusion chromatography (SEC) is used to detect antibodies or The antigen-binding portion, for example, the monomer present in the vedolizumab population, is a high molecular weight (HMW) monomer. ) The relative levels of aggregates and low molecular weight (LMW) degradation products can be determined. The SEC method is H This provides size-based separation of antibody monomers from MW species and LMW degradation products. Samples and reference standards were analyzed using a commercially available SEC column with appropriate buffer solutions. To obtain. For example, in some embodiments, SEC analysis is performed using a G3000 SWxl column ( Tosoh Bioscience, King of Prussia, PA (USA) ), or two G3000 SWxl columns connected in tandem, and isocrate This can be performed using a sodium phosphate-sodium chloride buffer system (pH 6.8). The elution of monomers is monitored at 280 nm. Main peak (monomer) and total peak. The area is evaluated to determine the purity. Sample purity (%) (calculated as monomer %), H MW aggregates (%) and / or LMW degradation products (%) have been reported.

[0319] In one embodiment, SEC analysis is performed using two G3000 SWxl devices connected in tandem. This involves injecting the sample into a ram, monitoring the elution of protein species at 280 nm, and Isocratic phosphate, in which the main peak (monomer) and total peak area are measured. The process is carried out using a salt-sodium chloride buffer system (pH 6.8).

[0320] IX. Pharmaceutical compositions and their use Compositions comprising vedolizumab provided herein, for example, basic isoform species A composition containing vedolizumab with reduced levels, and / or host cell protein levels Compositions containing vedolizumab with reduced efficacy may be incorporated into pharmaceutical formulations for therapeutic use. Pharmaceutical formulations containing vedolizumab can be prepared by any suitable method.

[0321] In one embodiment, a pharmaceutical formulation containing the composition described herein is a lyophilized pharmaceutical formulation. In one embodiment, the lyophilized preparation is stored in a single container, for example, a vial, as a single dose. It is possible. Containers, such as vials, may be refrigerated at approximately 2-8°C or at room temperature. For example, at approximately 20°C to 35°C, approximately 25°C, or approximately 30°C, it can be administered to the target that requires it. They are stored until they are used. Vials are available in, for example, 10, 20, or 50cc vials. It's possible. Containers, for example vials, contain approximately 90-115 mg, approximately 95-105 mg, and less. At least about 100mg, about 135-160mg, about 145-155mg, at least about 15 0 mg, approximately 180-220 mg, approximately 190-210 mg, approximately 195-205 mg, less Approximately 200 mg, approximately 280 mg to 320 mg, approximately 290 mg to 310 mg, and at least Approximately 300 mg, approximately 380-420 mg, approximately 390-410 mg, at least approximately 400 mg Approximately 580-620 mg, approximately 590-610 mg, or at least approximately 600 mg of anti-alpha It may also contain 4β7 antibody. In one embodiment, the vial contains approximately 200 mg of anti-α4β7 antibody. Includes. Vials contain, for example, approximately 100 mg, 108 mg, 150 mg, and 200 mg. Delivery of approximately 300 mg, 400 mg, or 600 mg of anti-α4β7 antibody, for example, It may contain a sufficient anti-α4β7 antibody to enable manufacturing, such as vedolizumab. The vial contains approximately 15%, 12%, 10%, or 8% more anti-α4 than the prescribed dose. It may contain β7 antibodies.

[0322] In some embodiments, the compositions described herein are administered in a liquid such as sterile water. It is formulated as a dried, lyophilized pharmaceutical preparation that can be reconstituted. The preparation may be administered by parenteral injection via one of the above routes. Intravenous injection is performed using sterile Isotonic saline, buffer solution, e.g., phosphate-buffered saline or Ringer's solution (lactic acid or dextrin). In some cases, injection may be performed by further dilution with a (Troth) solution.

[0323] In some embodiments, the compositions described herein are liquids suitable for subcutaneous administration to humans. It is formulated as a pharmaceutical product. In some embodiments, the anti-α4β7 antibody is administered by subcutaneous injection. Therefore, for example, approximately 2, 3, or 4 weeks after the start of treatment or after the third subsequent dose. For example, approximately 54 mg, 108 mg, or approximately 165 mg or approximately 216 mg per dose. It is administered by dose.

[0324] In another embodiment, the composition according to this specification comprises an anti-α4β7 antibody, for example, vedolizumab. The container, e.g., vial, syrup, is used until it is administered to the person who needs it. The stable liquid pharmaceutical composition is stored in a range or cartridge at approximately 2-8°C. In some embodiments, the stable liquid pharmaceutical composition of the anti-α4β7 antibody is approximately 0% to 5.0%. Contains aggregates of %, 0%~2%, ≤2%, ≤1%, ≤0.6%, or ≤0.5%. Syringe The container or cartridge may be a 1 mL or 2 mL container (for example, 160 mL) (In the case of g / mL doses) or, for example, higher doses (at least 320 mg or 40 If the dose is 0 mg or more, it may be 2 ml or more. The syringe or cartridge should be small. At least 20 mg, at least 50 mg, at least 70 mg, at least 80 mg g, at least about 100 mg, at least about 108 mg, at least about 120 mg, less At least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least approximately 300 mg, at least approximately 360 mg, at least approximately 400 mg It may contain g, or at least about 500 mg, of anti-α4β7 antibody. In some embodiments, The container, for example, a syringe or cartridge, contains approximately 20-120 mg, approximately 40 mg. 70 mg, approximately 45-65 mg, approximately 50-57 mg, or approximately 54 mg of anti-α4β7 antibody, For example, it may be manufactured to deliver vedolizumab. In other embodiments, a syringe or The cartridges contain approximately 90-120mg, 95-115mg, and 100-112mg. Alternatively, it may be manufactured to deliver approximately 108 mg of an anti-α4β7 antibody, such as vedolizumab. In other embodiments, the syringe or cartridge contains approximately 140-250 mg, approximately 1 50~200mg, approx. 160~170mg, approx. 160~250mg, approx. 175mg~21 0 mg, or approximately 160 mg, 165 mg, 180 mg, or 200 mg of anti-alpha-4 A β7 antibody, such as vedolizumab, can be manufactured to deliver this antibody.

[0325] Containers that can be used to store and freeze the purified compositions described herein include: Examples include recarbonate bottles (for IV formulations) or PETG bottles (for subcutaneous formulations). After dispensing the formulation into bottles, freezing may occur (for example, below -60 degrees Celsius). ).

[0326] A pharmaceutical composition is any vedolizumab composition provided herein, for example, a basic a Compositions containing vedolizumab with reduced levels of isoform species, and / or host cell cells Vedolizumab with reduced protein levels and a pharmaceutically acceptable carrier or excipient The composition may include the following: In some embodiments, the pH of the pharmaceutical composition is 6.0~ It is between 7.0, for example, pH 6.0-6.2, pH 6.0-6.4, pH 6.0-6. .6, pH6.0~6.8, pH6.1~6.3, pH6.1~6.5, pH6.1~6 .7, pH6.1~6.9, pH6.2~6.4, pH6.2~6.6, pH6.2~6 .8, pH6.2~7.0, pH6.3~6.5, pH6.3~6.7, pH6.3~6 .9, pH6.4~6.6, pH6.4~6.8, pH6.4~7.0, pH6.5~6 .7, pH6.5~6.9, pH6.6~pH6.8, pH6.6~7.0, pH6.7 The pH is ~6.9, or pH 6.8~7.0. In some embodiments, the pharmaceutical composition is , approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7 The pH is approximately 6.8, 6.9, or 7.0.

[0327] The pharmaceutical composition may be further supplemented with amino acids or sugars. In some embodiments... The pharmaceutical composition further contains amino acids such as arginine or histidine. In that embodiment, the pharmaceutical composition further comprises a sugar such as sucrose or trehalose. In some embodiments, the pharmaceutically acceptable compositions of anti-α4β7 antibodies provided herein are A subset of embodiments includes arginine, histidine, and / or polysorbate 80. The pharmaceutical composition of the anti-α4β7 antibody provided herein is citrate, arginine It contains histidine and / or polysorbate 80.

[0328] Compositions comprising vedolizumab provided herein, for example, basic isoform species A composition containing vedolizumab with reduced levels, and / or host cell protein levels Compositions containing vedolizumab with reduced integrin α4 It can be used in methods that inhibit β7 activity.

[0329] In one embodiment, the compositions described herein are effective in treating human diseases or disorders. The treatment involves administering a composition containing anti-α4β7 antibodies in a given amount to the target disease or disorder. It may be used for treatment. Human subjects include adults (e.g., 18 years of age or older), adolescents, or Children are acceptable. Human subjects may also be 65 years of age or older.

[0330] In one embodiment, the composition described herein is an immunomodulator, TNF-alpha-antagonism In cases of inadequate response, loss of response, or treatment with nistrone or a combination thereof. It is used to treat subjects who may have been intolerant to treatment. At the very least, they are receiving treatment with one corticosteroid (for example, prednisone). For example, a patient shows an insufficient response to corticosteroids for the treatment of inflammatory bowel disease, Possible tolerance or dependence. Inadequate response to corticosteroids. The response involves administering 30 mg of prednisone orally daily for two weeks, or intravenously for one week. Having a history of at least one 4-week induction regimen, including a dose equivalent to that given. Nevertheless, it refers to the signs and symptoms of persistent active illness. Loss of response means that corticosteroids are used in a manner equivalent to 10 mg of prednisone taken orally daily. This means that two attempts to gradually reduce the dose below a certain level have failed. This indicates corticosteroid intolerance. This includes Cushing's syndrome, osteopenia / osteoporosis, hyperglycemia, insomnia and / or infection. This medical history includes the following.

[0331] Immunomodulators include, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. It could be Xart. An inadequate response to an immunomodulator is at least one 8-week period. Regimen or oral azathioprine (1.5 mg / kg or more), 6-mercaptopurine ( (0.75 mg / kg or more), or methotrexate (12.5 mg / week or more) It refers to the signs and symptoms of a disease that persists despite its history. Immunomodulatory intolerance and While not limited to these, possible side effects include nausea / vomiting, abdominal pain, pancreatitis, LFT abnormalities, and lymphopenia. These include disease, TPMT gene mutations, and / or infection.

[0332] A TNFα antagonist is, for example, a substance that inhibits the biological activity of TNFα and also... For example, TNFα, monoclonal antibodies, for example, REMICADE (inflix) Simab, Humirah (adalimumab), Cimzia (certolizumab pegol), S Circulating receptor fusion drugs such as IMPONI (golimumab) or ENBREL (etanercept) It is a factor that binds to the synthetic protein. Insufficient response to TNF-α antagonists and The treatment involves administering infliximab 5 mg / kg (IV) twice, at least two weeks apart; Adali Administer 80 mg of mumab subcutaneously once, followed by 40 mg at least two weeks apart; Alternatively, 400 mg of certolizumab pegol administered subcutaneously in two doses at least two weeks apart. Despite having a history of at least one 4-week induction regimen, the patient continues to be active. This refers to the signs and symptoms of a disease. Loss of response to TNF-α antagonists is a previous state. This refers to the recurrence of symptoms during maintenance therapy following clinical benefit. However, this does not limit the causes of infusion-related reactions, demyelination, congestive heart failure, and / or infection. These are some examples.

[0333] For patients with ulcerative colitis, loss of maintenance of remission as used herein means at least A 3-point Mayo score and at least 2 modified Baron scores. This refers to an increase in the Baron Score.

[0334] In one embodiment, the diseases that can be treated accordingly are not limited to, Inflammatory bowel disease (IBD), for example, ulcerative colitis, Crohn's disease, ileitis, celiac disease, Non-tropical sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagen Pouchitis resulting from iosinophilic colitis, eosinophilic gastroenteritis, or ileal pouchitis following rectal and colectomy, and ileosalveolar ileum. Anastomosis is one example. In some embodiments, inflammatory bowel disease is Crohn's disease or ulcerative colitis. It is colitis. Additional diseases that can be treated include, for example, primary sclerosing cholangitis (PSC). ), and graft-versus-host disease (GVHD) are also mentioned.

[0335] Ulcerative colitis is a type of ulcerative colitis that is moderate to severely active (for example, subclinical endoscopy). The score may be 2 or 3, and the Mayo score may be 6-12. Treatment is moderate to severe. In patients suffering from active ulcerative colitis, the induction and maintenance of clinical response and clinical remission It may induce and maintain or lead to mucosal healing. Treatment also involves the patient's corticosteroids. This may result in a reduction, elimination, or reduction and elimination of costeroid use (for example, co (Remission without steroids)

[0336] Crohn's disease can also be moderate to severe active Crohn's disease (for example, Crohn's disease Disease Activity Index (CDAI) score: 220-450. Treatment involves moderate to severe activity of the disease. In patients suffering from Roon's disease, the goal is to achieve a clinical response or clinical remission. There is a possibility of this. Treatment also involves reducing, eliminating, or decreasing the patient's use of corticosteroids. It can also lead to elimination (for example, remission without corticosteroids).

[0337] Pancreatitis and insulin-dependent diabetes can be treated using the compositions of the present invention. It is a different disease. MAdCAM (e.g., MAdCAM-1) is a disease of NOD (non-obese diabetes). ) Several blood vessels of the exocrine pancreas from mice, as well as from BALB / c and SJL mice. It has been reported that it is expressed by MAdCAM (e.g., MAdCAM-1). It has been reported that the expression of this gene was induced in the endothelium of inflamed pancreatic islets in NOD mice. Furthermore, MAdCAM (for example, MAdCAM-1) is used in the early stages of islet inflammation to treat NOD pancreatic islet endothelium. It was the major adlessin expressed by (Hanninen, A., et al.) ., J. Clin. Invest., 92: 2509-2515 (1993)). NO. Treating D mice with anti-MAdCAM or anti-beta7 antibodies prevented the development of diabetes. (Yang et al., Diabetes, 46:1542-1547 (1997 Furthermore, accumulation of lymphocytes expressing α4β7 was observed within the pancreatic islets, and MAdCAM- 1 is related to the binding of lymphoma cells to blood vessels from inflamed pancreatic islets via α4β7 (Ha nninen, A., et al., J. Clin. Invest., 92: 2509 -2515 (1993)) or mantle cell lymphoma in the gastrointestinal tract (Geissmann et al., Am. J. Pathol., 153:1701-1705 (1998) They are related.

[0338] Examples of inflammatory diseases related to mucosal tissue that can be treated using the composition of the present invention and For example, cholecystitis, cholangitis (Adams and Eksteen Nature Rev. iews 6:244-251(2006) Grant et al., Hepatol ogy 33:1065-1072(2001)), for example, primary sclerosing cholangitis, for example Behcet's disease of the intestines, or pericholangitis (tissue surrounding the bile ducts and liver), and graft grafts. Primary disease (e.g., in the gastrointestinal tract (e.g., after bone marrow transplantation) (Petrovic et al .Blood 103:1542-1547(2004)) is an example. Crohn's disease As can be seen, inflammation often spreads beyond the mucosal surface, and therefore, sarcoidosis Chronic gastritis, such as autoimmune gastritis and other chronic inflammatory diseases (Katakai et al. l., Int.Immunol., 14:167-175 (2002)) and other idiopathic The condition may be suitable for treatment.

[0339] The present invention also relates to a method for inhibiting leukocyte infiltration in mucosal tissue. The present invention also relates to cancer The present invention relates to a method for treating (for example, α4β7-positive tumors such as lymphoma). Other examples of inflammatory diseases involving mucosal tissue that can be treated with the preparation include mastitis. Examples include mammary gland disease and irritable bowel syndrome.

[0340] The etiology utilizes the interaction between MAdCAM (e.g., MAdCAM-1) and α4β7. The disease or pathogen may be treated with the anti-α4β7 antibody in the formulation described herein. Examples of such diseases include immunodeficiency disorders caused by the human immunodeficiency virus. Harm can be cited (see, for example, WO2008140602).

[0341] The composition of the present invention contains an effective amount that inhibits the binding of α4β7 integrin to its ligand. It is administered as an anti-α4β7 antibody. In the case of treatment, the effective dose is the desired therapeutic (including preventive) effect. Sufficient to achieve (e.g., α4β7 integrin-mediated binding and / or signaling) Reduces or prevents leukocyte transmission, thereby hindering leukocyte adhesion and infiltration and / or related cell transmission. A sufficient amount to inhibit the response. An effective amount of anti-α4β7 antibody, e.g., α4β7 integrin Saturation of, for example, sufficient effective titer to maintain neutralization, is a clinical response in inflammatory bowel disease. Or it may induce remission. An effective amount of anti-α4β7 antibody may be effective in treating ulcerative colitis or Crohn's disease. It may lead to mucosal healing. The formulation of the present invention can be administered in a single dose or multiple doses. It may be administered in doses. The dosage may be determined by methods known in the art, e.g. For example, it may depend on the individual's age, sensitivity, tolerance, and overall health. Examples of dosage modes and This may involve local routes such as nasal, inhalation, or transdermal administration, or via a feeding tube or suppository. This can be administered via enteral routes such as , as well as intravenous, intramuscular, subcutaneous, intra-arterial, intraperitoneal, or intravitreous administration. Which parenteral routes are available? In one embodiment, the total dose is 165 mg. Another embodiment In one embodiment, the total dose is 108 mg. In another embodiment, the total dose is 216 mg. In this embodiment, the total dose is 300 mg.

[0342] In some embodiments, the diseases described herein, such as UC or Crohn's disease, are treated. The medication regimen for treatment has two stages: an induction phase and a maintenance phase. The antibody or its antigen-binding fragment is opposed to the antibody or its antigen-binding fragment. For specific purposes, such as inducing immune tolerance, or to induce a clinical response in inflammatory bowel disease To rapidly provide an effective amount of antibody or its antigen-binding fragment suitable for improving symptoms It is administered by the method provided. When the patient is first treated with anti-α4β7 antibody, treatment When treatment is administered shortly after, for example, after anti-α4β7 antibody therapy, more than 3 months, 4 If it exceeds a month, exceeds 6 months, exceeds 9 months, exceeds 1 year, exceeds 18 months When treatment is received more than two years later, or when symptoms of inflammatory bowel disease recur, for example, If there is a relapse after remission, even if induction therapy is administered during the maintenance phase of anti-α4β7 antibody therapy... Good. In some embodiments, the induction phase regimen is maintained during the maintenance regimen. An average trough serum concentration higher than the average steady-state trough serum concentration, for example, the following dose It brings up the concentration from the previous day.

[0343] During the maintenance phase, the antibody or its antigen-binding fragment maintains a stable level of antibody or its Administered in a manner that continues the response achieved by induction therapy using antigen-binding fragments. Maintenance regimens can prevent symptom relapses or relapses of inflammatory bowel disease. For example, the regimen may be a simple medication regimen, or it may be a rare treatment that is necessary for the treatment of a specific condition. It can provide convenience to the patient, such as being a top. In some embodiments, the maintenance regimen is For example, in the formulations described herein, low dose, infrequent administration, self-administration, and the aforementioned Anti-α4β7 antibody or This may include administration of antigen-binding fragments.

[0344] In one embodiment, for example, during the induction phase of treatment, the drug regimen is used to reduce inflammation in human patients. An effective amount of anti-α4β7 antibody in the formulation described herein for inducing remission of ventriculointestinal disease It provides an antigen-binding fragment. The induction period is approximately 4 weeks, 5 weeks, or 6 weeks. The treatment may last approximately 7 or 8 weeks. In some embodiments, the induction regimen This is due to high doses, frequent administration of anti-α4β7 antibodies or their antigen-binding fragments, and high Strategies selected from a group consisting of combinations of dose and frequency of administration are, for example, described herein. It may be used in the formulations described. Induction dosing may be one dose or two or more doses, for example. In that case, at least two doses may be administered. During the induction period, the dose is once a day, every other day, for one week. It can be administered twice a day, once a week, once every 10 days, once every two weeks, or once every three weeks. In some embodiments, the induction dose is administered within the first two weeks of treatment with an anti-α4β7 antibody. It is administered to the patient. In one embodiment, induction medication is administered once at the start of treatment (day 0) and during treatment. This may occur approximately once every two weeks after the start of the treatment. In another embodiment, the induction period is six weeks. In another embodiment, the induction period is 6 weeks, and multiple induction doses are administered during the first two weeks. It will be administered. In some embodiments, for example, when initiating treatment for a patient with severe inflammatory bowel disease... In combination (for example, in patients who have failed anti-TNFα therapy), the induction period is mild or moderate. It is necessary to have a longer duration than patients with the disease. In some embodiments, severe The induction period for patients with the disease is at least 6 weeks, at least 8 weeks, and at least 10 weeks. The interval may be at least 12 weeks, or at least 14 weeks. In one embodiment, For patients with severe illness, the induction regimen involves administration at week 0 (initiation of treatment). This may include administration at 2 weeks and administration at 6 weeks. Patients with severe illness may start treatment at week 0. This may include administration at the start of the course of treatment, administration at week 2, administration at week 6, and administration at week 10. .

[0345] This treatment is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks. It may be administered once every 8 weeks or once every 10 weeks. Higher or more frequent doses, e.g. For example, every other day, once a week, once every two weeks, once every three weeks, or once every four weeks, active disease To induce remission of a patient or to treat a new patient, for example, an anti-α4β7 antibody It may be useful in inducing remission for [the condition]. Once every two weeks, once every three weeks, once every four weeks Dosage once, once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 10 weeks is recommended. In preventative therapy, for example, it may be useful for maintaining remission in patients with chronic diseases. In one embodiment, The treatment regimen is as follows: day 0, approximately week 2, approximately week 6, and then every one or two weeks thereafter. It is a treatment. In certain cases, the treatment regimen is day 0, approximately week 2, approximately week 6, and so on. The treatment is then administered every 8 weeks. In another aspect, the induction treatment regimen consists of a total of 6 treatments. The treatment is performed every other day.

[0346] In some embodiments, permanent clinical remission, e.g., 6 months or 1 year after the start of treatment. During that period, at least two, at least three, and at least four visits by the doctor providing care. Sustained clinical remission throughout the course can be achieved with an optimized dosing regimen.

[0347] In some embodiments, a sustained clinical response, for example, for at least 6 months after the start of treatment. Clinical responses lasting at least 9 months and at least 1 year are achieved with an effective medication regimen. It is possible.

[0348] This disclosure will be further illustrated by the following embodiments. The embodiments shown are for illustrative purposes only. Therefore, this disclosure should not be interpreted as limiting the scope or content in any way. . [Examples]

[0349] The following examples illustrate an exemplary process for purifying vedolizumab from CHO cell cultures. This explains the various steps involved in the process.

[0350] Example 1. Control of charged isoforms of vedolizumab Vedolizumab has three charged isoforms: acidic, primary, and basic. On-exchange (CEX)-HPLC is used to chromatograph acidic, major, and basic species. Based on the relative area of ​​the lamb, the isoform distribution of vedolizumab can be quantified. Figure 1 shows exemplary CEX-HPLC profiles illustrating the three vedolizumab species.

[0351] Using CEX-HPLC, the charged isoform distribution of vedolizumab was analyzed across various Evaluation was performed after storage under the specified conditions. As summarized in Table 1, the manufacturing process of vedolizumab Within the range of retention, the distribution of charged isoform species is affected, and basic isoforms are retained. I was most affected by that incident. [Table 1]

[0352] These results show that the proportion of different isoforms of vedolizumab is different under certain conditions, which can lead to antibody It has been shown that it can be predictably regulated by maintaining basic isophores. The number of species tends to increase during retention periods at pH levels below approximately 6.5. In contrast, basic conditions... Isoform species decrease during retention periods at pH levels above approximately 6.5. These changes are Observations were observed in vedolizumab manufactured at both pilot scale (Table 2) and production scale (Table 3). It can be inferred. [Table 2] [Table 3]

[0353] To evaluate the charge isoform distribution of vedolizumab derived from GS-CHO cells Additional experiments were conducted. The proportion of the major acidic and basic species of vedolizumab varied depending on the p H(that is, pH is 4.7, 5.1, 5.3, 5.7, 5.9, 6.1, 6.5, and The results were evaluated after storage at 5°C or room temperature for 0-7 days as described in 6.9). As shown in Table 4-11, Improved stability was observed at pH levels of 5.9 or higher. Following the capture and elution of protein A... The typical pH range for neutralization (pH approximately 4.9-5.2) is associated with increased formation of basic species. When vedolizumab was stored at pH 5.9 or 6.1, storage at pH below 5.9 Compared to the basic species observed during the organism's existence, the increase in basic species was slower. See Tables 10 and 11. As shown, the increase of basic chemical species stops at pH 6.5 or higher, or conversely, increases. Enaru. [Table 4] [Table 5] [Table 6]

Table 7

Table 8

Table 9

Table 10

Table 11

[0354] This data shows that the levels of the major isoforms and basic isoform species in the vedolizumab preparation can be regulated by pH. In particular, the basic isoform species increase upon exposure of the antibody to low pH (<pH 5.9), and concomitantly, the major isoform decreases. The level of the basic isoform species increases more rapidly and to a greater extent upon exposure to a decrease in pH. Furthermore, this trend is reversible upon exposure to an increase in pH, for example, >pH 6.5.

[0355] Example 2. Reduction of the basic isoform of vedolizumab To further evaluate the effect of pH on the formation of the basic isoform of vedolizumab, the antibody was exposed to high pH conditions ( 200 mM Tris, pH 9), and the isoform distribution of vedolizumab was quantified using cation exchange (CEX)-HPLC. For each condition, the relative areas under the chromatogram peaks corresponding to the acidic, major, and basic isoforms ​​​The relative amount of each vedolizumab isoform was quantified by determining the area.

[0356] As shown in Table 12, three peaks corresponding to the basic species of vedolizumab are at pH 6.3. (Control) was present (i.e., "basic peak 1", "basic peak 2", and "basic Peak 3). As shown in Table 12, increasing the pH resulted in a significant decrease in basic peak 2. They figured it out. [Table 12] We collected substances eluted from CEX resin that have a characteristic retention time for basic peak 2, and enzymes. After decomposition, the sample was analyzed using mass spectrometry (MS). A characteristic MS-P of succinimide was observed. The basic peak 2 CEX fraction of the control preparation was present, but exposure to pH 9 was... It was not present in the preparations analyzed later. Analysis of the primary amino acid sequence of vedolizumab revealed , a residue favorable for isomerization of aspartic acid to succinimide, i.e., the n+1 position. [ka] Vedolizumab CDR-H3 aspartate, located near glycine or serine. The residue was identified. This discovery led to an increase in the basic species of vedolizumab observed at low pH. The main reason is that this may be due to the isomerization of the aspartic acid residue to succinimide. This suggests that maintaining the antibody at or near a neutral pH delays the formation of basic peak 2. To reverse or prevent the isomerization reaction, raising the pH (>pH 6.9) during treatment can reverse the reaction. The succinimide can be converted back to aspartic acid (or isoaspartic acid).

[0357] To further evaluate the effect of pH on basic peak 2, the antibody was subjected to various pH conditions. After exposure to (pH 6.5, pH 7, pH 8, pH 8.5, or pH 9), CEX- The relative peak area was determined by HPLC. As shown in Table 13, the basic peak 2 is It is highly sensitive to pH, decreasing with increasing pH, which is consistent with the results reported in Example 1. They are doing it. [Table 13]

[0358] Next, the levels of the vedolizumab isoform corresponding to basic peak 2 were compared with two different levels. Vedolizumab preparations derived from CHO cell lines (DHFR-CHO and GS-CHO) The evaluation was based on the following: As shown in Table 14, DHFR-CHO cell lines and GS-CHO cell lines A decrease in basic peak 2 was observed in vedolizumab preparations derived from both sources. [Table 14]

[0359] Example 3. Influence of anion exchanger loading conductivity on host cell protein clearance. sound Reducing the amount of host cell protein contaminants in therapeutic protein compositions is generally done. Therefore, to produce a vedolizumab composition with reduced host cell protein content, We investigated the manufacturing method.

[0360] Standard operating range of buffer conductivity for anion exchange (AEX) (e.g., anion exchange Q membrane absorption) The radiation level (through the attachment device) is approximately 11-15 mS / cm (average approximately 13.6 mS / cm). To evaluate impurity clearance under conditions exceeding the quasi-operating range, use A with lower conductivity. Impurity clearance was tested in compositions obtained using EX conditions. Two independent initial preparations of the clarified harvest were tested (harvest 1 and harvest 2). The conductivity of the terial can be set to standard conductivity (approximately 13.6 mS / cm) or low conductivity (approximately 11 mS / cm). After adjusting to ( / cm), all samples were subjected to AEX purification. As shown in Table 15, HCP The level is lower compared to standard conductivity AEX load material. It decreased due to alcohol. [Table 15]

[0361] Next, the AEX capability (flow-through mode) is defined under standard operating conditions (i.e., 13-1 Test using a loading buffer with a conductivity range lower than 5 mS / cm. Table 16 shows the loading conditions, conductivity, host cell protein content, and acidity. This summarizes the proportions of basic isoforms. The test involved CHO host cell proteins. The procedure was performed using a cellulose ELISA. As shown in Table 16, the amount of host cell protein This decreased along with the decrease in the conductivity of the AEX loading buffer. [Table 16]

[0362] Next, we examined the effect of the decrease in AEX conductivity on HCP clearance using two different HCPs. Evaluation was performed by ELISA. The first method used rabbit primary antibody and anti-rabbit secondary antibody. This was used with HCP ELISA (RaRa). In the second method, HCP ELISA Rabbit primary antibodies and goat secondary antibodies were used in (RaGo). Tables 17 and 18 show the results. These two HCPELISA methods after using standard or low conductivity AEX loads. This summarizes the results. Both methods reduce the conductivity of the AEX load material. This indicates that HCP clearance is improved by doing so. [Table 17] [Table 18]

[0363] The amount of HCP in recombinant protein preparations is variable. As shown herein, HCP clearance from dolizumab preparations is reduced during purification using AEX. This can be improved by lowering the conductivity. Regardless of the HCP concentration in the starting material, the conductivity is low. Under AEX conditions, a significant reduction in HCP was achieved compared to standard high-conductivity AEX conditions. It was done.

[0364] Example 4. Effect of equilibration and washing conditions on the loading capacity of mixed mode resin To minimize vedolizumab loss during purification, various purification conditions were investigated. We identified the step in which the yield of antibody products decreases under high-concentration loading conditions. Protein loss occurs during the washing step of the purification of black hydroxyapatite (CHT). This was observed. For example, Figure 2 shows a standard washing and equilibration buffer (75 mM N Use aCl, 10 mM sodium phosphate, pH 7.2) at a dose of 27 mg / ml or 35 mg / ml. Comparing the elution profiles of vedolizumab from CHT columns after loading mg / ml. They are comparing. As shown in Figure 2, during the washing step under high-concentration loading conditions, The protein content in the column gradually began to decrease (35 mg / ml).

[0365] Increase the loading capacity of vedolizumab into the CHT column and reduce loss during the CHT washing step. To improve the yield of vedolizumab by reducing the amount of protein that is broken down, alternative C HT equilibrium, loading, and wash buffer were evaluated. Vedolizumab on CHT column The loading volume (g / l) and the yield obtained were measured in a standard high-pH CHT buffer. Using 75 mM NaCl, 10 mM sodium phosphate, pH 7.2, the CHT column Compared to the operation, a low pH buffer (50 mM NaCl, 10) is used for equilibrating and washing CHT. This was confirmed after operating a CHT column using mM sodium phosphate (pH 6.7). Figure 3 As shown, during the washing step using standard CHT buffer conditions (dashed line), a considerable amount of liquid was lost. Although a certain amount of protein was lost, when using a low pH buffer (solid line), the protein loss was reduced. No loss was observed. Furthermore, as summarized in Table 19, the equilibration and washing steps Using a low pH buffer in the process allows for an increase in the amount of material loaded onto the CHT column. This allows for the standard elution to be performed without changing the quality of the product in the final eluate (e.g., aggregate percentage). A yield 2–3% higher was obtained compared to that achieved using standard CHT buffer. [Table 19]

[0366] Equal portions Although the present invention has been described in connection with its detailed description, the foregoing description limits the scope of the present invention. It is not intended to show, but not to limit, that it is defined in the attached claims. It should be understood that other aspects, advantages, and modifications are within the scope of the attached claims.

[0367] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. In addition, materials, methods, and examples are illustrative and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of the present invention, but preferred methods and materials are described herein. The present invention includes the following embodiments. [1] A method for producing a composition containing vedolizumab, the following: To provide a composition containing vedolizumab at a pH higher than pH 6.5; The composition containing vedolizumab is incubated for at least 20 minutes to 10 hours; This allows for the production of a composition containing vedolizumab, The method, including the method described above. [2] The method according to [1] above, wherein the method is a method for producing a composition comprising vedolizumab in which the level of basic vedolizumab isoform species is reduced, and the method is according to [1] above, wherein the method is a method for producing a composition comprising vedolizumab in which the level of basic vedolizumab isoform species is reduced. [3] The method according to [1] or [2] above, wherein the method produces a composition comprising a vedolizumab having <16%, <15%, <14%, <13%, <12%, <11%, or <10% basic vedolizumab isoform species. [4] The method according to any one of the above [1] to [3], wherein the incubation is performed during vedolizumab purification, and the incubation is performed (a) before ultrafiltration / diafiltration (UF / DF) of the antibody, or (b) before formulation of the antibody in a pharmaceutically acceptable buffer. [5] The method according to any one of the above [1] to [4], wherein the incubation is performed at ambient temperature. [6] The method according to any of the above [1] to [4], wherein the incubation is carried out at 15 to 30°C, or the incubation is carried out at 20 to 25°C. [7] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is provided at a pH of about 6.5 to 8.5. [8] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 to 8.0. [9] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 to 7.5.

[10] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is provided at a pH of approximately pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, or pH 8.5.

[11] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 10 to 120 hours.

[12] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 120 hours.

[13] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 96 hours.

[14] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 72 hours.

[15] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 48 hours.

[16] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of at least 12 hours.

[17] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 120 hours.

[18] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 96 hours.

[19] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 72 hours.

[20] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 48 hours.

[21] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is incubated for a period of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours.

[22] A method for purifying a humanized anti-α4β7 antibody or its antigen-binding portion from a harvested cell culture, (i) To provide harvested clarified cell cultures obtained from cultures of recombinant host cells expressing the anti-α4β7 antibody or its antigen-binding moiety, and (ii) Purifying the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture, wherein the antibody is exposed to a pH of 4.0 or less for 24 hours or less, and the purification is performed. The method wherein the anti-α4β7 antibody or its antigen-binding portion includes a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence described in SEQ ID NO: 5.

[23] The method according to

[22] , wherein the anti-α4β7 antibody or its antigen-binding moiety has a reduced level of basic isoform species (determined by CEX) compared to a control, the control being a composition comprising the anti-α4β7 antibody or its antigen-binding moiety produced by the same method, and the antibody is exposed to a pH of 4.0 or less (e.g., pH 3.6 to 4.0) for a longer period, i.e., more than 24 hours.

[24] The method according to

[22] or

[23] , wherein the anti-α4β7 antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion.

[25] The method according to any of the above embodiments, wherein the composition comprising vedolizumab or its antigen-binding moiety comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2), and the method produces a composition comprising vedolizumab or its antigen-binding moiety with a reduced level of BP2.

[26] The method according to

[25] above, wherein the method produces a composition comprising vedolizumab or its antigen-binding moiety and having less than 2%, less than 1.5%, less than 1%, or less than 0.7% of BP2.

[27] The method according to any of the above embodiments, wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

[28] The method according to

[27] above, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

[29] The method according to

[28] , wherein the CHO cell culture comprises CHO cells lacking dihydrofolate reductase (DHFR) expression.

[30] The method according to

[28] above, wherein the CHO cell culture comprises CHO cells lacking glutamine synthetase (GS) expression.

[31] The method according to any of the above embodiments, further comprising purifying the composition comprising vedolizumab from mammalian host cell proteins (HCPs) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

[32] The method according to

[31] , wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

[33] The method according to

[32] above, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

[34] The method according to

[32] above, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

[35] The method according to

[31] above, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

[36] The method according to

[35] , wherein the composition comprising vedolizumab is purified by cation exchange chromatography prior to the incubation.

[37] The method according to

[35] , wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

[38] The method according to

[31] above, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

[39] The method according to

[38] , wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

[40] The method according to

[38] , wherein the composition comprising vedolizumab is purified using anion exchange chromatography after the incubation.

[41] The method according to

[31] above, wherein the composition comprising vedolizumab is purified using CHT chromatography.

[42] The method according to

[41] above, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

[43] The method according to

[41] above, wherein the composition comprising vedolizumab is purified using CHT chromatography after the incubation.

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

[43] , comprising incorporating the composition into a pharmaceutical preparation.

[45] The method according to

[44] above, wherein the pharmaceutical preparation is a freeze-dried pharmaceutical preparation.

[46] The method according to

[45] above, wherein the freeze-dried pharmaceutical preparation is a dried freeze-dried pharmaceutical preparation.

[47] The method according to

[46] , further comprising the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid so that it is suitable for administration.

[48] The method according to

[44] above, wherein the pharmaceutical preparation is a liquid pharmaceutical preparation.

[49] A composition comprising vedolizumab, wherein the composition is produced by the method described in any of [1] to

[48] above.

[50] A composition comprising vedolizumab, which can be obtained by any of the methods described in [1] to

[48] above.

[51] The composition according to

[49] or

[50] , wherein the basic vedolizumab isoform species comprises less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the vedolizumab species present in the composition.

[52] A low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the total basic isoform species of the anti-α4β7 antibody, wherein the basic isoform species has a net positive charge compared to the major isoform of the anti-α4β7 antibody and can be quantified by determining the relative area of ​​the peak that elutes more slowly from a cation exchange (CEX) resin than the peak corresponding to the major isoform, and the anti-α4β7 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 5.

[53] The composition according to

[52] above, wherein the composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2).

[54] The composition according to

[53] above, wherein the composition contains less than 2% BP2.

[55] The composition according to

[53] above, wherein the composition contains less than 1.5% BP2.

[56] The composition according to

[53] above, wherein the composition contains less than 1% BP2.

[57] The composition according to

[53] above, wherein the composition contains less than 0.7% BP2.

[58] The composition according to

[53] above, wherein the ratio of BP1 to BP2 is at least 3.

[59] The composition according to

[53] above, wherein the ratio of BP1 to BP2 is at least 5.

[60] The composition according to

[53] above, wherein the ratio of BP1 to BP2 is at least 7.

[61] The composition according to

[53] above, wherein the ratio of BP1 to BP2 is at least 10.

[62] A pharmaceutical composition comprising any of the compositions described in

[52] to

[61] above, and a pharmaceutically acceptable carrier or excipient.

[63] A method for producing a composition containing vedolizumab, the following: (a) Contacting a sample containing vedolizumab and host cell protein (HCP) with an anion exchange resin in the presence of a loading buffer, wherein the conductivity of the loading buffer is 11 mS / cm or less so that the HCP binds to the anion exchange resin; (b) Collecting the flow-through material from the anion exchange resin, The method wherein the eluent contains vedolizumab.

[64] The method according to

[63] , wherein the method is a method for producing a composition comprising vedolizumab having a reduced amount of HCP, and the flow-through material comprises vedolizumab and a reduced amount of HCP.

[65] The method according to

[63] or

[64] , wherein the loading buffer has a conductivity of 9 mS / cm to 11 mS / cm.

[66] The method according to

[63] or

[64] , wherein the loading buffer has a conductivity of 10 mS / cm or less.

[67] The method according to

[63] or

[64] , wherein the loading buffer has a conductivity of 9 mS / cm or less.

[68] The method according to

[63] or

[64] , wherein the loading buffer has a conductivity of approximately 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm, or 11 mS / cm.

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

[63] to

[68] , wherein the HCP is Chinese hamster ovary (CHO) cell protein.

[70] The method according to

[69] , wherein the HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression.

[71] The method according to

[69] above, wherein the HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

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

[63] to

[71] , further comprising contacting the anion exchange resin with a washing buffer.

[73] The method according to

[72] , wherein the washing buffer has an conductivity of less than 11 mS / cm.

[74] The method according to

[72] , wherein the washing buffer has an conductivity of 9 mS / cm to 11 mS / cm.

[75] The method according to

[72] above, wherein the washing buffer has the same conductivity as the loading buffer.

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

[63] to

[75] , wherein the loading buffer comprises sodium chloride and / or sodium phosphate.

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

[63] to

[75] , wherein the washing buffer comprises sodium chloride and / or sodium phosphate.

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

[63] to

[77] , wherein the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

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

[63] to

[78] , wherein the anion exchange resin contains a quaternary amine functional group.

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

[63] to

[79] , wherein the sample comprising vedolizumab and HCP is derived from a mammalian cell culture after one or more chromatographic separation steps.

[81] The method according to

[80] , wherein the one or more chromatographic separation steps include one or more steps selected from the group consisting of affinity chromatography, cation exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

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

[63] to

[81] , wherein the amount of HCP in the flow-through material is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm or less, 4.5 ppm or less, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, or 2 ppm or less.

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

[63] to

[82] , wherein the amount of HCP in the flow-through material is reduced by at least 50% compared to the amount of HCP in the flow-through material produced when the method is carried out using a loading buffer with a conductivity of 12 mS / cm or more and the same sample.

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

[63] to

[83] , further comprising treating the flow-through material to replace the elution buffer by a process comprising ultrafiltration and / or diafiltration into a buffer containing one or more pharmaceutically acceptable carriers or excipients.

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

[63] to

[84] , comprising incorporating the composition into a pharmaceutical preparation.

[86] The method according to

[85] above, wherein the pharmaceutical preparation is a freeze-dried pharmaceutical preparation.

[87] The method according to

[86] above, wherein the freeze-dried pharmaceutical preparation is a dried freeze-dried pharmaceutical preparation.

[88] The method according to

[87] , further comprising the step of reconstituting the dried, lyophilized pharmaceutical formulation with a liquid so that it is suitable for administration.

[89] The method according to

[85] above, wherein the pharmaceutical preparation is a liquid pharmaceutical preparation.

[90] The method according to

[89] or

[48] above, wherein the liquid pharmaceutical preparation is suitable for subcutaneous administration to humans.

[91] A composition comprising vedolizumab produced by any of the methods described in

[63] to

[90] above.

[92] The composition comprises vedolizumab, which can be obtained by any of the methods described in

[63] to

[90] above.

[93] The composition according to

[91] or

[92] , wherein the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm or less, 4.5 ppm or less, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, or 2 ppm or less.

[94] A method for increasing the yield of vedolizumab recovered after elution from a mixed-mode chromatography resin, comprising: equilibrating the mixed-mode chromatography resin with an equilibration buffer; loading a solution containing vedolizumab and a loading buffer onto the mixed-mode chromatography resin so that vedolizumab binds to the mixed-mode chromatography resin; washing the mixed-mode chromatography resin with a washing buffer; and eluting vedolizumab from the mixed-mode chromatography resin with an elution buffer, wherein the pH of the equilibration buffer, the loading buffer and / or the washing buffer is 7.0 or less.

[95] The method according to

[94] , wherein the pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.0 to 7.0.

[96] The method according to

[94] , wherein the pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.5 to 7.0.

[97] The method according to

[94] , wherein the pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.6 to 6.8.

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

[94] to

[97] , wherein the salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 30 mM to 70 mM.

[99] The method according to

[98] , wherein the salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 40 mM to 70 mM.

[0100] The method according to

[98] , wherein the salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 50 mM to 65 mM.

[0101] The method according to

[98] , wherein the salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 55 mM to 65 mM.

[0102] The method according to any one of the above

[98] to

[0101] , wherein the salt comprises sodium chloride and / or sodium phosphate.

[0103] The method according to any one of the above

[94] to

[97] , wherein the sodium chloride concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 30 mM to 70 mM.

[0104] The method according to

[0103] above, wherein the sodium chloride concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 40 mM to 70 mM.

[0105] The method according to

[0103] above, wherein the sodium chloride concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 40 mM to 60 mM.

[0106] The method according to

[0103] above, wherein the sodium chloride concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 45 mM to 55 mM.

[0107] The method according to

[0103] , wherein the sodium chloride concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is approximately 50 mM.

[0108] The method according to any one of the above

[0103] to

[0107] , wherein the equilibration buffer, the loading buffer, and / or the washing buffer further comprises sodium phosphate.

[0109] The method according to any one of the above

[94] to

[0108] , wherein the pH of the equilibration buffer, the loading buffer, and / or the washing buffer are the same.

[0110] The method according to any one of the above

[94] to

[0109] , wherein the salt concentrations of the equilibration buffer, the loading buffer, and / or the washing buffer are the same.

[0111] The method according to any one of the above

[94] to

[0108] , wherein the equilibration buffer, the loading buffer, and / or the washing buffer are the same buffer.

[0112] The method according to any one of the above

[94] to

[0111] , wherein the mixed mode resin is a ceramic hydroxyapatite resin.

[0368] Table 20

Claims

1. A method for obtaining a composition comprising a purified humanized anti-α4β7 antibody or its antigen-binding moiety from a harvested cell culture, (i) A step of providing a harvested product of a clarified cell culture obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody or its antigen-binding moiety, and (ii) A step of purifying the anti-α4β7 antibody or its antigen-binding portion from the harvested cell culture to obtain a purified composition, wherein the antibody is exposed to a pH of 4.6 to 5.5 for at least 2 hours and no more than 24 hours, the step comprising: Here, the anti-α4β7 antibody or its antigen-binding portion includes a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence described in SEQ ID NO:

5. The purified composition comprises less than 16% of the anti-α4β7 antibody or a basic isoform species of its antigen-binding moiety (determined by cation exchange (CEX) HPLC), The recombinant host cell is a CHO cell. The aforementioned method.

2. The method according to claim 1, wherein the anti-α4β7 antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion.

3. The method according to claim 1 or 2, wherein the purified composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2), and the purified composition comprises a reduced level of BP2.

4. The method according to claim 3, wherein the purified composition contains less than 2%, less than 1.5%, less than 1%, or less than 0.7% of BP2 (determined by CEX HPLC).

5. The method according to any one of claims 1 to 4, wherein the culture of CHO cells comprises CHO cells lacking dihydrofolate reductase (DHFR) expression or CHO cells lacking glutamine synthetase (GS) expression.

6. The method according to any one of claims 1 to 5, further comprising the step of purifying the composition comprising the anti-α4β7 antibody or its antigen-binding moiety from mammalian host cell proteins (HCPs) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

7. The method according to claim 6, wherein the composition comprising the anti-α4β7 antibody or its antigen-binding moiety is purified using an affinity chromatography resin containing protein A.

8. The method according to any one of claims 1 to 7, wherein the purified composition contains less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the basic isoform species of the humanized anti-α4β7 antibody or its antigen-binding moiety (determined by CEX HPLC).

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