Method for purifying antibody

Abstract

A method for purifying an antibody, comprising the steps of: preparing a solution containing an antibody; and purifying the antibody by filtering the solution using a porous membrane comprising regenerated cellulose. A method for purifying an antibody, comprising the steps of: preparing a solution containing an antibody; and purifying the antibody by filtering the solution using a porous membrane comprising regenerated cellulose, (A) the Tr-Tm of the antibody in hydrophobic interaction chromatography being 15 minutes or more and / or (B) the k value of the antibody in hydrophobic interaction chromatography being 19 or more.

Description

Technical Field

[0001] The present invention relates to a method for purifying antibodies. Background Art

[0002] As a measure to improve the safety against viruses, biological preparations such as plasma fractionation preparations and biopharmaceuticals derived from human blood are introduced into their production processes for virus removal / inactivation steps. Among them, the virus removal method based on filtration using a porous hollow fiber membrane is an effective method for reducing viruses without denaturing useful proteins (for example, see Patent Documents 1 to 3).

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: International Publication No. 2015 / 156401

[0006] Patent Document 2: International Publication No. 2017 / 170874

[0007] Patent Document 3: International Publication No. 2022 / 118943 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] As useful proteins used in biological preparations, antibodies can be cited. In addition, multispecific antibodies that have multiple antigen binding sites and are specific to multiple antigens have attracted attention in recent years. Including multispecific antibodies, there are antibodies that are easy to clog filters and are difficult to filter. Therefore, one of the problems of the present invention is to provide a purification method for antibodies that can purify antibodies that are difficult to filter.

[0010] Solutions for solving problems

[0011] [1] The antibody purification method of the embodiment comprises the following steps: preparing a solution containing an antibody; and purifying the antibody by filtering the solution using a porous membrane containing regenerated cellulose, wherein the antibody in the solution contains a particle size distribution of 22.0 nm or more. Here, the particle size distribution can be measured by dynamic light scattering.

[0012] [2] In the method for purifying an antibody according to [1] above, the porous membrane comprising regenerated cellulose may be a porous hollow fiber membrane.

[0013] [3] In the porous film containing regenerated cellulose of the above-mentioned [1] or [2], the elastic limit pressure may be 200 kPa or more.

[0014] [4] In the method for purifying an antibody according to any one of [1] to [3] above, the particle size distribution of the antibody in the solution containing the antibody may include a range of 24.0 nm or more.

[0015] [5] In the method for purifying an antibody according to any one of [1] to [4] above, the antibody may include antibody aggregates.

[0016] [6] In the method for purifying an antibody according to any one of [1] to [5] above, the antibody may be an antibody capable of binding to two or more different antigens.

[0017] [7] In the method for purifying an antibody according to any one of [1] to [6] above, the antibody may be a monoclonal antibody.

[0018] [8] In the method for purifying an antibody according to any one of [1] to [7] above, the antibody may be contained in an antibody-drug conjugate.

[0019] [9] In the method for purifying an antibody according to any one of [2] to [8] above, the ratio (R / t) of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t) may be 8.4 or less.

[0020]

[10] In the method for purifying an antibody according to any one of [2] to [9] above, the membrane thickness (t) of the porous hollow fiber membrane may be in the range of 20 μm to 70 μm.

[0021]

[11] In the method for purifying an antibody according to any one of [1] to

[10] above, the regenerated cellulose may be regenerated cellulose obtained by a cuprammonia method.

[0022]

[12] In the method for purifying an antibody according to any one of [2] to

[11] above, the pore size of the inner surface of the porous hollow fiber membrane may be larger than the pore size of the outer surface.

[0023]

[13] In the method for purifying an antibody according to any one of [2] to

[12] above, the porous hollow fiber membrane may have an inclined structure in which the pore size decreases from the inner surface side toward the outer surface side.

[0024]

[14] In the antibody purification method described in any one of [2] to

[13] above, the water permeability of the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37° C. may be 10 L / (m 2 ·hr) and above and 50L / (m 2 hr) or less.

[0025]

[15] In the method for purifying an antibody according to any one of [2] to

[14] above, the bubble point of the porous hollow fiber membrane may be 1.2 MPa or higher.

[0026]

[16] In the method for purifying an antibody according to any one of [1] to

[15] above, viruses may be removed during the process of purifying the antibody.

[0027]

[17] In the method for purifying an antibody according to any one of [2] to

[16] above, the parvovirus removal rate (LRV) of the porous hollow fiber membrane may be 4.0 or higher.

[0028]

[18] In the method for purifying an antibody according to any one of [1] to

[17] above, the pH of the solution containing the antibody may be 4.5 to 10.0.

[0029]

[19] In the method for purifying an antibody according to any one of [1] to

[18] above, the salt concentration of the solution containing the antibody may be 0 mmol / L or more and 1000 mmol / L or less.

[0030]

[20] In the method for purifying an antibody according to any one of [1] to

[19] above, the conductivity of the solution containing the antibody may be 0 mS / cm or more and 100 mS / cm or less.

[0031]

[21] An antibody purification method according to one embodiment comprises the following steps: preparing a solution containing an antibody; and purifying the antibody by filtering the solution using a porous membrane containing regenerated cellulose, wherein (A) the Tr-Tm of the antibody in hydrophobic interaction chromatography is greater than 15 minutes and / or (B) the k value of the antibody in hydrophobic interaction chromatography is greater than 19. Here, Tr is the time it takes for the antibody to pass through the hydrophobic interaction chromatography column in the hydrophobic interaction chromatography device and reach the detector. Tm is the time it takes for the antibody to reach the detector in the hydrophobic interaction chromatography device with the hydrophobic interaction chromatography column removed. The k value is provided by the following formula. It should be noted that T0 is the time it takes for the antibody to directly pass through the hydrophobic interaction chromatography column in the hydrophobic interaction chromatography device and reach the detector without interacting with the hydrophobic interaction chromatography column.

[0032] k=(Tr-T0) / (T0-Tm)

[0033]

[22] In the method for purifying an antibody of

[21] above, the porous membrane comprising regenerated cellulose may be a porous hollow fiber membrane.

[0034]

[23] In the method for purifying an antibody according to

[21] or

[22] , the elastic limit pressure of the porous membrane comprising regenerated cellulose may be 200 kPa or more.

[0035]

[24] In the method for purifying an antibody according to any one of

[21] to

[23] above, Tr-Tm may be 16 minutes or more and / or k value may be 20 or more.

[0036]

[25] In the method for purifying an antibody according to any one of

[21] to

[24] above, the antibody may be an antibody capable of binding to two or more different antigens.

[0037]

[26] In the method for purifying an antibody according to any one of

[21] to

[25] above, the antibody may be a monoclonal antibody.

[0038]

[27] In the method for purifying an antibody according to any one of

[21] to

[26] above, the antibody may be contained in an antibody-drug conjugate.

[0039]

[28] In the method for purifying an antibody according to any one of

[22] to

[27] above, the ratio (R / t) of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t) may be 8.4 or less.

[0040]

[29] In the method for purifying an antibody according to any one of

[22] to

[28] above, the membrane thickness (t) of the porous hollow fiber membrane may be in the range of 20 μm to 70 μm.

[0041]

[30] In the method for purifying an antibody according to any one of

[21] to

[29] above, the regenerated cellulose may be regenerated cellulose obtained by a cuprammonia method.

[0042]

[31] In the method for purifying an antibody according to any one of

[22] to

[30] above, the pore size of the inner surface of the porous hollow fiber membrane may be larger than the pore size of the outer surface.

[0043]

[32] In the method for purifying an antibody according to any one of

[22] to

[31] above, the porous hollow fiber membrane may have an inclined structure in which the pore size decreases from the inner surface side toward the outer surface side.

[0044]

[33] In the antibody purification method described in any one of

[22] to

[32] above, the water permeability of the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37° C. may be 10 L / (m 2 ·hr) and above and 50L / (m 2 hr) or less.

[0045]

[34] In the method for purifying an antibody according to any one of

[22] to

[33] above, the bubble point of the porous hollow fiber membrane may be 1.2 MPa or higher.

[0046]

[35] In the method for purifying an antibody described in any one of

[21] to

[34] above, viruses may be removed during the process of purifying the antibody.

[0047]

[36] In the method for purifying an antibody according to any one of

[22] to

[35] above, the parvovirus removal rate (LRV) of the porous hollow fiber membrane may be 4.0 or higher.

[0048]

[37] In the method for purifying an antibody according to any one of

[21] to

[36] above, the pH of the solution containing the antibody may be 4.5 or more and 10.0 or less.

[0049]

[38] In the method for purifying an antibody according to any one of

[21] to

[37] above, the salt concentration of the solution containing the antibody may be 0 mmol / L or more and 1000 mmol / L or less.

[0050]

[39] In the method for purifying an antibody according to any one of

[21] to

[38] above, the conductivity of the solution containing the antibody may be greater than or equal to 0 mS / cm and less than or equal to 100 mS / cm.

[0051] Effects of the Invention

[0052] According to the present invention, a method for purifying an antibody capable of purifying an antibody that is difficult to filter can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of a membrane cross section of a porous hollow fiber membrane according to an embodiment, showing the relationship between the inner diameter (R) and the membrane thickness (t) of the porous hollow fiber membrane.

[0054] Figure 2 It is a graph showing the particle size distribution of antibodies.

[0055] Figure 3 This is a graph showing the retention time of antibodies when using a hydrophobic interaction chromatography column.

[0056] Figure 4 is a graph showing the filtration rate of a solution containing an antibody.

[0057] Figure 5 is a graph showing the filtration rate of a solution containing an antibody.

[0058] Figure 6 is a graph showing the filtration rate of a solution containing an antibody.

[0059] Figure 7 is a graph showing the filtration rate of a solution containing an antibody.

[0060] Figure 8 is a graph showing the filtration rate of a solution containing an antibody.

[0061] Fig. 9 is a graph showing the filtration rate of a solution containing an antibody.

[0062] Fig.10 is a graph showing the filtration rate of a solution containing an antibody.

[0063] Fig.11 is a graph showing the filtration rate of a solution containing an antibody.

[0064] Fig.12 is a graph showing the filtration rate of a solution containing an antibody.

[0065] Fig.13 is a graph showing the filtration rate of a solution containing an antibody.

[0066] Fig.14 It is a graph showing the particle size distribution of antibodies.

[0067] Fig.15 is a graph showing the filtration rate of a solution containing an antibody. DETAILED DESCRIPTION

[0068] The present invention will be described in detail below in conjunction with a specific embodiment (hereinafter referred to as "this embodiment"). However, the present invention is not limited to the following embodiment and can be implemented in any manner within the scope of the present invention.

[0069] The antibody purification method of the present embodiment comprises the following steps: preparing a solution containing an antibody; and, filtering the solution using a porous membrane containing regenerated cellulose to purify the antibody. As the form of the porous membrane, specifically, a porous hollow fiber membrane, a porous flat membrane, a nonwoven fabric, a woven fabric, any form is acceptable. In addition, the elastic limit pressure of the porous membrane can be 200 kPa or more.

[0070] In a solution containing an antibody, the particle size of the antibody can be distributed. The particle size distribution of the antibody can be measured using a dynamic light scattering (DLS) method. The details of the dynamic light scattering method will be described in the embodiments. The particle size distribution of the antibody obtained using the dynamic light scattering method described in the embodiments can include a range of more than 22.0 nm, a range of more than 23.0 nm, a range of more than 24.0 nm, a range of more than 25.0 nm, or a range of more than 26.0 nm. The upper limit of the particle size distribution of the antibody can be 50 nm, 40 nm, 35 nm, or 30 nm. The particles providing a large particle size in the particle size distribution can be monomers of the antibody or aggregates of the antibody.

[0071] In addition, as an example of an index representing the characteristics of the antibody to be purified, the hydrophobicity degree of the antibody can be cited. The hydrophobicity degree of the antibody can be measured by the length of the time Tr that passes through the hydrophobic interaction chromatographic column and arrives at the detector. The higher the hydrophobicity of the antibody, the longer the time Tr. The time Tr may be affected by the length of the piping of the hydrophobic interaction chromatographic device, but the time Tm of the detection peak obtained when the hydrophobic interaction chromatographic column is removed from Tr can eliminate the influence of the length of the piping. When using the assay method described in detail in the embodiment, the value of the Tr-Tm of the antibody contained in the solution to be purified in this embodiment can be more than 15 minutes, more than 16 minutes, more than 17 minutes, more than 18 minutes or more than 19 minutes. In addition, the value of the Tr-Tm of the antibody can be less than 40 minutes, less than 35 minutes, less than 30 minutes or less than 25 minutes.

[0072] In addition, as an index indicating the hydrophobicity of the antibody that is not affected by the piping length of the chromatographic device or the measurement conditions, the retention coefficient k value described in detail in the Examples can be used. The k value of the antibody contained in the solution purified in this embodiment obtained by the analysis method described in the Examples can be 19 or more, 19.5 or more, or 20 or more. In addition, the k value of the antibody can be 30 or less, 28 or less, or 25 or less.

[0073] The solution containing antibodies may contain antibody aggregates. Antibody aggregates contain antibodies of dimer or higher. Generally, antibody aggregates have more charge than antibody monomers. In addition, antibody aggregates are generally more hydrophobic than antibody monomers. When the salt concentration of the solution increases, the hydrophobic interactions between antibodies become stronger, and there is a tendency to easily produce antibody associations and aggregates. In addition, there is a tendency for the aggregation of antibodies to increase when the temperature of the solution increases.

[0074] The antibody can be a human antibody or an antibody protein derived from mammals other than humans, such as cows and mice. Alternatively, the antibody can also be a chimeric antibody protein with human IgG and a humanized antibody. A chimeric antibody with human IgG refers to an antibody whose variable region is derived from organisms other than humans such as mice, but the other constant regions are replaced with immunoglobulins derived from humans. In addition, a humanized antibody refers to an antibody whose complementarity-determining region (CDR) in the variable region is derived from organisms other than humans, but the other framework regions (FR) are derived from humans. Compared with chimeric antibodies, humanization further reduces immunogenicity.

[0075] The category (isotype) and subtype of the antibody are not particularly limited. For example, antibodies can be divided into five types, namely IgG, IgA, IgM, IgD and IgE, according to the structural differences in the constant region. Among them, the antibody that is the object of purification of the porous hollow fiber membrane of the embodiment can be any of the five categories. In addition, there are four subtypes of IgG in human antibodies, namely IgG1 to IgG4, and there are two subtypes of IgA, namely IgA1 and IgA2. The subtype of the antibody that is the object of purification of the porous hollow fiber membrane of the embodiment can be any kind. It should be noted that antibody-related proteins such as Fc fusion proteins in which the Fc region is bound to proteins are also included in the antibodies that are the objects of purification of the porous hollow fiber membrane of the embodiment.

[0076] Antibodies can also be classified according to their sources. The antibodies to be purified by the porous hollow fiber membrane of the embodiment can be any of natural human antibodies, recombinant human antibodies manufactured by genetic recombination technology, monoclonal antibodies or polyclonal antibodies. Antibodies can be derived from plasma products, or from cell culture fluids, for example. In the case of obtaining antibodies by cell culture, animal cells or microorganisms can be used as cells. As animal cells, the types are not particularly limited, and CHO cells, Sp2 / 0 cells, NS0 cells, Vero cells, PER.C6 cells, etc. can be listed. As microorganisms, the types are not particularly limited, and Escherichia coli, yeast, etc. can be listed.

[0077] Antibodies can be multispecific antibodies that are specific for a variety of antigens. Multispecific antibodies are sometimes referred to as next-generation antibodies. As examples of multispecific antibodies, full-length antibodies, antibodies with more than two VL and VH domains, DART (registered trademark, MacroGenics, Inc.) molecules, Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies (TandAb), and three-chain antibodies and antibody fragments connected by covalent or non-covalent bonds can be cited.

[0078] A multispecific antibody that is specific for two antigens is called a bispecific antibody. A bispecific antibody can specifically bind to two different epitopes on one biological molecule. Alternatively, a bispecific antibody can specifically bind to epitopes on two different biological molecules.

[0079] The antigen-binding domain of a bispecific antibody comprises, for example, two VH / VL units, the first VH / VL unit specifically binds to the first epitope, and the second VH / VL unit specifically binds to the second epitope. Each VH / VL unit comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0080] The VH / VL unit comprises at least one H chain hypervariable region (VH HVR) and at least one L chain hypervariable region (VL HVR). The VH / VL unit comprises, for example, one, two or all three VH HVRs and one, two or all three VL HVRs. The VH / VL unit may further comprise at least a portion of a framework region. The VH / VL unit may comprise three VH HVRs and three VL HVRs, in which case the VH / VL unit comprises one, two, three or all four H chain framework regions and one, two, three or all four L chain framework regions.

[0081] A VH / VL unit that also contains at least a portion of a heavy chain constant region and / or at least a portion of a light chain constant region is also referred to as a half antibody. A half antibody contains at least a portion of a single heavy chain variable region and at least a portion of a single light chain variable region. A half antibody is also defined as a monovalent antigen-binding polypeptide. A half antibody may also contain a constant region.

[0082] For example, a half-antibody comprises a portion of the heavy chain variable region sufficient to form an intramolecular disulfide bond with another half-antibody. In addition, for example, a half-antibody comprises a hole mutation or a knob mutation that can heterodimerize with another half-antibody containing a complementary hole mutation or knob mutation. Here, a mutation that introduces a protuberance (knob) into a polypeptide is referred to as a knob mutation. A mutation that introduces a cavity (hole) into a polypeptide is referred to as a hole mutation.

[0083] For example, a bispecific antibody comprising two half antibodies and binding to two antigens comprises a first half antibody that binds to the first epitope but not to the second epitope and a second half antibody that binds to the second epitope but not to the first epitope.

[0084] Bispecific antibodies are, for example, knob-in-hole (KiH) antibodies. Knob-in-hole refers to the association of two polypeptides by introducing a protrusion (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where they interact. The knob-in-hole can be introduced into, for example, the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody. For example, bispecific antibodies can be produced by pairing two different heavy chains through the knob-in-hole. In addition, bispecific antibodies are, for example, CrossMab antibodies. CrossMab refers to the association of two different antibodies, each with its own half.

[0085] In multispecific antibodies, multiple L chains may be common. By modifying different L chains for multiple antigens to produce a common L chain, and co-expressing it with multiple different H chains, an antibody having a common L chain and maintaining the ability to bind to multiple antigens can be produced.

[0086] When the hydrophobicity of an antibody is high, it is usually prone to aggregation and association. When the hydrophobicity of a multispecific antibody is high, it is also prone to aggregation and association. However, even if the hydrophobicity of a multispecific antibody is low, it is also prone to aggregation and association. Although not bound by theory, it is believed that multispecific antibodies have multiple different antigen-binding sites, so the charge distribution within the antibody molecule is asymmetric. Therefore, the charge interaction between antibody molecules becomes stronger, and aggregation and association are prone to occur.

[0087] Therefore, in a solution containing a multispecific antibody, the particle size distribution of the antibody may include a range of 22.0 nm or more. In addition, the Tr-Tm of the multispecific antibody in hydrophobic interaction chromatography may be 15 minutes or more. In addition, the k value of the multispecific antibody may be 19 or more.

[0088] The antibody may be included in an antibody-drug conjugate (ADC). In the case of an antibody-drug conjugate, a drug may be linked to the antibody. Examples of drugs linked to the antibody include low molecular weight drugs, such as anticancer agents.

[0089] The solvent used in the solution containing the antibody can be pure water or a buffer. The buffer is not particularly limited, and examples thereof include a buffer in which at least any one of tris salt, acetate, Tween, sorbitol, maltose, glycine, arginine, lysine, histidine, sulfonate, phosphate, citric acid and sodium chloride is dissolved. The concentration of the salt contained in the buffer is not particularly limited as long as it can dissolve the antibody. The lower limit of the salt concentration of the buffer depends on the type of buffer, for example, 0 mmol / L or more, 0.5 mmol / L or more, 1.0 mmol / L or more, 5.0 mmol / L or more, 10.0 mmol / L or more, 15.0 mmol / L or more, or 25.0 mmol / L or more. The upper limit of the salt concentration of the buffer solution depends on the type of the buffer solution, and is, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 400 mmol / L or less, 300 mmol / L or less, or 200 mmol / L or less.

[0090] The pH of the buffer solution is not particularly limited. The lower limit of the pH of the buffer solution depends on the type of the buffer solution, and is, for example, 4.5 or more, 5.0 or more, 5.5 or more, or 6.0 or more. The upper limit of the pH of the buffer solution depends on the type of the buffer solution, and is, for example, 10.0 or less, 9.0 or less, 8.0 or less, 8.5 or less, 8.0 or less, 7.5 or less, or 7.0 or less.

[0091] The conductivity of the buffer solution is not particularly limited. The lower limit of the conductivity of the buffer solution depends on the type of the buffer solution, for example, 0 mS / cm or more, 1 mS / cm or more, 2 mS / cm or more, 3 mS / cm or more, 4 mS / cm or more, or 5 mS / cm or more. The upper limit of the conductivity of the buffer solution depends on the type of the buffer solution, for example, 100 mS / cm or less, 90 mS / cm or less, 80 mS / cm or less, 70 mS / cm or less, 60 mS / cm or less, 50 mS / cm or less, 40 mS / cm or less, 30 mS / cm or less, or 20 mS / cm or less.

[0092] The antibody concentration in the solution is not particularly limited as long as the antibody is dissolved in the solution. The lower limit of the antibody concentration in the solution is, for example, more than 0.01 mg / mL, more than 0.05 mg / mL, more than 0.10 mg / mL, more than 0.50 mg / mL, more than 1.00 mg / mL or more than 5.00 mg / mL. The upper limit of the antibody concentration in the solution is, for example, less than 100 mg / mL, less than 90 mg / mL, less than 80 mg / mL, less than 70 mg / mL, less than 60 mg / mL, less than 50 mg / mL, less than 40 mg / mL, less than 30 mg / mL, less than 25 mg / mL or less than 20 mg / mL.

[0093] The porous hollow fiber membrane of the present embodiment is a hollow membrane having a porous structure with many pores for permeating or capturing substances. The shape of the porous hollow fiber membrane is not particularly limited, and may have a cylindrical continuous shape. In this specification, the surface located inside the cylinder of the porous hollow fiber membrane is recorded as the inner surface, and the surface located outside the cylinder is recorded as the outer surface.

[0094] The porous membrane of the present embodiment is not particularly limited as long as it is a porous membrane containing regenerated cellulose. The regenerated cellulose is not particularly limited as long as it is cellulose obtained by dissolving natural cellulose by chemical treatment, forming a stock solution, and then regenerating it by other chemical treatment. Examples thereof include regenerated cellulose obtained by a method of preparing it from a cuprammonium cellulose solution (cuprammonium method) or a method of preparing it by saponifying cellulose acetate with an alkali (saponification method).

[0095] The porous film of the present embodiment may contain components other than regenerated cellulose, and a part of the regenerated cellulose may also be modified. Examples may be given of regenerated cellulose in which cellulose hydroxyl is modified by esterification or partially cross-linked regenerated cellulose, etc. In addition, the porous film surface may be coated with a polymer film. As the polymer for coating, examples may be given of copolymers of polyhydroxyethyl methacrylate, 2-hydroxyethyl methacrylate and acrylamide, polymethoxyethyl acrylate, 2-hydroxyethyl methacrylate and diethylaminoethyl methacrylate, copolymers of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate, copolymers of 2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate and n-butyl methacrylate, hydroxypropyl cellulose, polyvinyl pyrrolidone, or copolymers of vinyl pyrrolidone and vinyl acetate, etc.

[0096] The elastic limit pressure of the porous flat membrane can be defined as: when the surface side of the flat membrane having a layer with an equal or larger pore size than the other surface is pressurized with air, the change of the filtration flow rate deviates from the linear change as the pressure increases, and the filtration flow rate is no longer the same as the filtration flow rate before the above operation when the flat membrane after the above operation is pressurized again with a pressure below it. The deviation of the filtration flow rate of the flat membrane from the linear change occurs due to the change in pore size caused by the plastic deformation of the flat membrane. Here, when the liquid flows into the flat membrane by pressurization, the filtration flow rate of the flat membrane can be calculated by dividing the weight of the liquid passing through the flat membrane by the time elapsed from the start of filtration. It is preferred that the virus removal and water permeability of the porous flat membrane in various integrity tests such as inspections, filtration and leakage tests in the flat membrane manufacturing process do not change substantially before and after the test, and the pressure used in the test is preferably selected as a pressure below the elastic limit. It should be noted that the elastic limit pressure of the porous flat membrane of the present embodiment is measured in a state where the porous flat membrane is wetted with water.

[0097] The elastic limit pressure of the porous hollow fiber membrane can be defined as: the pressure observed when the expansion deviates from the linear change due to the change in the outer diameter of the hollow fiber membrane accompanied by the pressure increase when air is pressurized from the inner surface side of the hollow fiber membrane. The expansion of the hollow fiber membrane deviates from the linear change due to the plastic deformation of the hollow fiber membrane. It is preferred that the virus removal and water permeability of the porous hollow fiber membrane do not substantially change before and after the test in various integrity tests such as inspections, filtration and leakage tests within the manufacturing process of the porous hollow fiber membrane, and the pressure used in the test is preferably selected below the elastic limit. It should be noted that the elastic limit pressure of the porous hollow fiber membrane of the present embodiment is measured when the porous hollow fiber membrane is wetted with water.

[0098] The elastic limit pressure of porous membrane is more than 200kPa, and can be more than 210kPa, more than 220kPa, more than 230kPa, more than 240kPa or more than 250kPa. Alternatively, the elastic limit pressure of porous hollow fiber membrane can be more than 215kPa, more than 225kPa, more than 235kPa, more than 245kPa, more than 255kPa, more than 270kPa or more than 280kPa. The upper limit value of the elastic limit pressure of porous hollow fiber membrane is not particularly limited as long as the pressure that can be actually applied, and can illustrate less than 1000kPa, less than 900kPa, less than 800kPa, less than 700kPa, less than 600kPa, less than 500kPa, less than 450kPa, less than 400kPa, less than 350kPa or less than 300kPa.

[0099] Compared with the film with low elastic limit pressure, the porous film with elastic limit pressure of more than 200kPa can accelerate the filtration speed. In addition, the filtration amount per unit time can be increased. And then, due to the good filtration efficiency, the membrane area can also be reduced, and the manufacturing cost can be reduced. Moreover, the porous film with elastic limit pressure of more than 200kPa is no longer necessary in the integrity test such as leakage test, and the evaluation test using colloidal gold is no longer necessary.

[0100] By using a porous membrane comprising regenerated cellulose having an elastic limit pressure of more than 200kPa, it is possible to filter a solution comprising antibodies that was previously thought to be difficult to filter due to blockage. The above-mentioned particle size distribution includes antibodies in the range of more than 22.0nm, antibodies with Tr-Tm of more than 15 minutes in the hydrophobic interaction chromatography, and antibodies with k values ​​of more than 19, which belong to antibodies that were previously thought to be difficult to filter due to blockage. The porous membrane comprising regenerated cellulose having an elastic limit pressure of more than 200kPa can filter antibodies in the range of more than 22.0nm in the particle size distribution, antibodies with Tr-Tm of more than 15 minutes in the hydrophobic interaction chromatography, and antibodies with k values ​​of more than 19 in the case of suppressing blockage.

[0101] The inter-membrane pressure difference during filtration is set high. In addition to the economic advantage of increasing the processing volume per unit time, it also has the advantage that the higher the inter-membrane pressure difference during filtration is set for the virus removal membrane, the greater the physical restriction force of the virus on the membrane, and thus the reliability of virus capture is improved. The inter-membrane pressure difference is preferably set to less than about 75% of the elastic limit pressure of the porous membrane. Therefore, the preferred inter-membrane pressure difference during filtration is 165kPa or more, 188kPa or more, or 225kPa or more. In addition, as another mode of the inter-membrane pressure difference during filtration, 150kPa or more, 200kPa or more, or 250kPa or more can be exemplified. The upper limit of the membrane pressure difference during filtration is not particularly limited as long as it is the pressure that can actually be applied, and can be exemplified as below 1000kPa, below 900kPa, below 800kPa, below 700kPa, below 600kPa, below 500kPa, below 450kPa, below 400kPa, below 350kPa or below 300kPa. The membrane pressure difference is treated synonymously with the filtration pressure of low pressure filtration under the condition of not applying particularly high filtration discharge pressure. In addition, as the control means of the membrane pressure difference, the filter object can be pressed and sent in the mode of fixed pressure with the pressure applied to the porous membrane, and the filter object can also be filtered at a fixed speed within the range of the elastic limit pressure that will not exceed the porous membrane.

[0102] Here, the differential pressure across the porous flat membrane refers to the pressure difference between the pressure on the inflow fluid side of the porous flat membrane and the pressure on the outflow fluid side of the porous flat membrane. Therefore, the differential pressure across the porous flat membrane is, for example, a value obtained by subtracting the pressure on the outflow fluid side of the porous flat membrane from the pressure on the inflow fluid side of the porous flat membrane.

[0103] In addition, the membrane pressure difference of the porous hollow fiber membrane refers to the pressure difference between the pressure on the inner surface side of the porous hollow fiber membrane and the pressure on the outer surface side of the porous hollow fiber membrane. Therefore, the membrane pressure difference of the porous hollow fiber membrane is, for example, a value obtained by subtracting the pressure on the outer surface side of the porous hollow fiber membrane from the pressure on the inner surface side of the porous hollow fiber membrane.

[0104] The porous hollow fiber membrane of this embodiment preferably has a ratio (R / t) of the inner diameter (R (μm)) to the membrane thickness (t (μm)) of 8.4 or less. Figure 1 As shown in the figure, the inner diameter (R) and the membrane thickness (t) are measured from a cross-sectional image of a hollow fiber cut into a disc in a dry state, the inner diameter is the inner surface diameter of the hollow fiber, and the membrane thickness is the vertical distance between the inner surface and the outer surface of the hollow fiber. Unless otherwise specified, the inner diameter (R) and the membrane thickness (t) represent the values ​​measured in a dry state.

[0105] Based on the curve of the porous hollow fiber membrane, the inventors of the present invention found that for a porous hollow fiber membrane containing regenerated cellulose suitable for a filter object with a particle size of less than 20nm to about 100nm, R / t, which is the ratio of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t), has a specific correlation with the elastic limit pressure. From the viewpoint of achieving an elastic limit pressure of more than 200kPa of the porous hollow fiber membrane, the upper limit value of R / t is preferably less than 8.4. Corresponding to the more preferred lower limit value of the above-mentioned elastic limit pressure, the more preferred range of R / t is less than 8.0, and the further preferred range is less than 7.7. From the viewpoint of stably producing the hollow fiber shape and satisfying the balance between the supply flow rate and the permeation flow rate as a hollow fiber filtration membrane, the lower limit value of R / t is preferably greater than 2.0, that is, the inner diameter is more than 2 times the membrane thickness.

[0106] The thickness of the porous flat membrane is preferably in the range of 20 μm or more and 100 μm or less. From the perspective of the convenience of designing an area for capturing microscopic substances using a sieve effect as a porous hollow fiber membrane, the membrane thickness is preferably 20 μm or more. In addition, from the perspective of the simplicity of setting the permeability of the porous hollow fiber membrane to a high level, the membrane thickness is preferably set to 100 μm or less. The membrane thickness of the porous hollow fiber membrane is more preferably in the range of 30 μm or more and 80 μm or less, and further preferably in the range of 40 μm or more and 70 μm or less.

[0107] The membrane thickness of the porous hollow fiber membrane is preferably in the range of 20 μm or more and 70 μm or less. From the perspective of the convenience of designing a region for capturing microscopic substances using a sieve effect as a porous hollow fiber membrane, the membrane thickness is preferably 20 μm or more. In addition, from the perspective of the simplicity of setting the permeability of the porous hollow fiber membrane to a high level, the membrane thickness is preferably set to 70 μm or less. The membrane thickness of the porous hollow fiber membrane is more preferably in the range of 30 μm or more and 60 μm or less, and more preferably in the range of 40 μm or more and 50 μm or less.

[0108] From the viewpoint of achieving a high flow rate and suppressing the clogging of the porous flat membrane, when filtering the antibody solution, it is preferred that the pore size of the surface of the porous flat membrane on the side where the antibody solution flows in is larger than the pore size of the surface on the side where the antibody solution flows out. Furthermore, from the viewpoint of improving the capture performance of the particles to be removed and suppressing the influence of clogging, it is more preferred to have an inclined structure in which the pore size decreases along the direction of travel of the antibody solution and also include a homogeneous structure with little pore size change to capture the particles to be removed. Here, the pore size refers to the size of the hole portion in the image obtained by observing the two surfaces of the flat membrane or the cross section formed by vertically cutting the surface of the flat membrane with an optical microscope or a scanning electron microscope, and the degree of difference obtained by the comparison is preferably clear enough to be visually recognized using a microscope image.

[0109] From the viewpoint of achieving high flow rate and suppressing clogging of porous hollow fiber membrane, it is preferred that the pore size of the inner surface of the porous hollow fiber membrane is larger than the pore size of the outer surface. Furthermore, from the viewpoint of improving the capture performance of the particles of the removal object and suppressing the influence of clogging, it is more preferred to have an inclined structure in which the pore size decreases from the inner surface side to the outer surface side and also includes a homogeneous structure with little pore size change to capture the particles of the removal object. Here, the pore size refers to the size of the hole portion in the image obtained by observing the inner surface, outer surface of the membrane or the cross-section of the hollow fiber membrane cut into discs with an optical microscope or a scanning electron microscope, and the degree of difference obtained by the comparison is preferably clear to the extent that it can be visually recognized using a microscope image.

[0110] From the viewpoint of achieving both the porous structure and excellent hydrophilicity required of a virus removal membrane, the porous membrane preferably includes regenerated cellulose obtained by the cuprammonia method. An example of a method for producing a porous hollow fiber membrane of the present embodiment using the cuprammonia method is described below.

[0111] First, a spinning solution having a cellulose concentration of 6% to 8% by mass, an ammonia concentration of 4% to 5% by mass, and a copper concentration of 2% to 3% by mass is prepared by dissolving cellulose in a copper ammonia solution, an aqueous solution having an acetone concentration of 30% to 50% by mass and an ammonia concentration of 0.5% to 1.0% by mass, i.e., an internal coagulation solution, and an aqueous solution having an acetone concentration of 20% to 40% by mass and an ammonia concentration of 0.2% or less by mass, i.e., an external coagulation solution are prepared. From the viewpoint of adjusting the speed of microphase separation of the spinning solution, an inorganic salt such as sodium sulfate may be contained in the range of about 0.03% to 0.1% by mass.

[0112] Next, the spinning solution is preferably ejected from the annular double-layer spinneret at a rate of 2 mL / min to 5 mL / min, and the internal coagulation solution is ejected from the central spinning outlet disposed in the central part of the annular double-layer spinneret at a rate of 0.3 mL / min to 3.0 mL / min. For example, in order to obtain a hollow fiber inner diameter and membrane thickness that achieves an elastic limit pressure exceeding 200 kPa, and in order to make the membrane thickness of the manufactured porous hollow fiber membrane within the preferred range of 20 μm to 70 μm, it is more preferred to set the solution ejection rate to 2.5 mL / min to 4 mL / min, and the internal coagulation solution rate to 0.3 mL / min to 1.6 mL / min. A further preferred method is to make the internal coagulation solution rate within the range of 0.3 mL / min to 1.4 mL / min. The spinning solution and the internal coagulation solution ejected from the annular double-layer spinneret are immediately immersed in the external coagulation solution, and after the internal coagulation solution and the external coagulation solution are coagulated, the film is wound up using a spinning frame.

[0113] The impregnation of the spinning solution and the internal coagulation solution into the external coagulation solution may include a method of impregnating the spinning solution and the internal coagulation solution into the external coagulation solution accumulated in the coagulation bath; a method of coagulating while flowing and falling together with the external coagulation solution in the spinning funnel; and a method using a U-shaped capillary as a method using the spinning funnel. From the viewpoint of achieving a membrane structure with a high particle removal rate by suppressing stretching during the coagulation process, the method using the U-shaped capillary is preferred.

[0114] From the viewpoint of forming the porous hollow fiber membrane into a membrane structure that stably achieves the water permeability and virus removal performance described later, the temperature of the external coagulation liquid is preferably controlled at a predetermined temperature selected from the range of 25° C. to 45° C. A more preferred temperature range is 30° C. to 45° C., and a further preferred range is 35° C. to 45° C.

[0115] The wound hollow fiber membrane is immersed in a 2% to 10% by mass aqueous solution of dilute sulfuric acid, and then washed with pure water to regenerate the cellulose. The water content of the hollow fiber membrane is replaced with an organic solvent such as methanol or ethanol. The two ends of the hollow fiber membrane bundle are fixed, and the hollow fiber membrane is stretched by 1% to 8%, and then dried under reduced pressure at 30°C to 60°C and below 5kPa to obtain a porous hollow fiber membrane in a dry state.

[0116] When filtering a solution containing an antibody using a porous hollow fiber membrane, in order to effectively capture particles of the object to be removed in the solution, it is preferred to use the porous hollow fiber membrane in a method of filtering the solution in a direction of flowing from the inner surface side of the hollow fiber toward the outer surface side (internal pressure filtration method). The particles of the object to be removed are, for example, viruses. Examples of viruses include parvoviruses, which are small viruses.

[0117] When using a porous hollow fiber membrane to remove viruses such as parvovirus, it is preferred that the water permeability of the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37°C is 10 L / (m 2 ·hr) and above and 50L / (m 2 ·hr) or less. The water permeability is the flow rate per unit time when filtering water by the internal pressure filtration method. When a virus removal membrane designed with a high water permeability is used, the virus removal process of biological agents can be carried out in a short time. On the other hand, the water permeability is a scale representing the overall average pore size of the porous hollow fiber membrane, and is designed to correspond to the size of the virus particles to be removed. Therefore, from the perspective of making the capture performance of small viruses less than 20nm in diameter more reliable, the water permeability is preferably 10L / (m 2 ·hr) and above and 50L / (m 2 ·hr) or less, more preferably 15L / (m 2·hr) and above and 45L / (m 2 Here, the water permeability is specified under the conditions of filtration pressure 27 kPa and 37° C. because these are the measurement conditions for the water permeability when the average pore size (nm) of a porous hollow fiber membrane is calculated in the technical field.

[0118] The water permeability of the porous hollow fiber membrane at a filtration pressure of 98 kPa and 25°C is preferably 20 L / (m 2 ·hr) and above and 100L / (m 2 ·hr) or less, more preferably 30L / (m 2 ·hr) and above and 85L / (m 2 hr) or less.

[0119] The bubble point of the porous hollow fiber membrane is, for example, above 1.2 MPa. Here, the bubble point refers to the scale of the size of the largest pores of the porous hollow fiber membrane. From the perspective of making the capture of parvoviruses less than 20 nm in diameter more practical, the lower limit of the bubble point is preferably above 1.3 MPa, more preferably above 1.4 MPa, and further preferably above 1.5 MPa. In addition, from the perspective of achieving the above-mentioned water permeability, the upper limit of the bubble point is preferably below 2.4 MPa, more preferably below 2.3 MPa, and further preferably below 2.2 MPa. It should be noted that the bubble point refers to: a test assembly that can seal one end of the porous hollow fiber membrane and pressurize it from the other end using air or nitrogen, and the gas flow rate leaked when the test assembly is immersed in a fluorine-based liquid with low surface tension and pressurized is 2.4 mL / min.

[0120] The virus removal performance of the porous membrane can be measured by comparing the 50% tissue culture infection value (TCID 50 The virus removal rate was evaluated by the logarithmic reduction value (LRV), which is the logarithmic reduction value of the ratio of 1:1 to 2:1.

[0121] When using porous hollow fiber membrane to remove parvovirus, 6.0TCID 50 / mL and above 8.0TCID 50 / mL or less of the stock solution containing parvovirus with a transmembrane pressure difference of 196kPa and 150L / m 2 The parvovirus removal rate when filtering a large amount of the sample is preferably 4.0 or more. Furthermore, considering the response to a large filtration volume and the influence of filtration pressure fluctuations, the parvovirus removal rate under the same conditions is more preferably 4.5 or more, and further preferably 5.0 or more. The LRV is preferably measured in the LRV measurement method described in (5) of the measurement method example of the embodiment described later using the virus-containing protein solution described in (5-A).

[0122] The longer the time of filtering with porous membrane, the more the filtration amount. Filtration time is, for example, more than 30 minutes, more than 1 hour, more than 3 hours or more than 6 hours. The upper limit of filtration time is not particularly limited, for example, less than 7 days, less than 6 days, less than 5 days, less than 4 days or less than 3 days.

[0123] Before the solution containing the antibody is purified by a porous membrane, other means can be used to purify or concentrate the solution containing the antibody. As examples of other means, methods using protein A carriers, affinity chromatography carriers, ion exchange chromatography carriers, hydrophobic interaction chromatography carriers, mixed mode chromatography carriers, hydroxyapatite, depth filters, ultrafiltration membranes, pre-filters and activated carbon can be cited. In addition, after the solution containing the antibody is purified by a porous hollow fiber membrane, the solution containing the antibody can be purified or concentrated by means such as described above. It should be noted that the method for purifying the solution containing the antibody by a porous hollow fiber membrane can be connected to the means such as described above and implemented continuously, or it can be implemented independently of the method such as described above.

[0124] Example

[0125] The present invention is described in more detail below by way of examples, but the present invention is not limited to the following examples and can be implemented in any manner without departing from the spirit of the present invention.

[0126] [Example of measurement method]

[0127] (1) Method for determining the elastic limit pressure of porous hollow fiber membranes

[0128] One end of a porous hollow membrane of 50 mm in length is sealed with a curable liquid resin such as polyurethane resin in a manner that air will not leak out, and the other end is bonded and fixed with a curable liquid resin such as polyurethane resin in a manner that the hollow portion will not be buried under the state of being inserted into a micro-coupler (made by Nitto Koki Co., Ltd., MC-04PH), and a measuring assembly is prepared. In addition, a pressurizing device equipped with a micro-coupler (made by Nitto Koki Co., Ltd., MC-10SM) is prepared in a manner that a pressure regulating valve, a pressure gauge, and a micro-coupler of a measuring assembly can be connected to a pipe for compressed air supply. The measuring assembly is connected to the pressurizing device in a state immersed in water, and compressed air is supplied to the hollow portion by increasing the pressure at intervals of 20 kPa, and the outer diameter of the hollow fiber at this time is measured using a size measuring device (made by KEYENCE CORPORATION, model LS-9006M). The outer diameter change rate (%) caused by each measuring pressure is calculated using the following formula, and a graph is prepared in which the X-axis is set to the measuring pressure (kPa) and the Y-axis is set to the outer diameter change rate (%).

[0129] Outer diameter change rate (%) = (D / D0-1) × 100

[0130] (Wherein, D: outer diameter at each pressure (μm), D0: initial value of outer diameter in non-pressurized state (μm))

[0131] Next, using 5 measured values ​​at 20 kPa intervals from 20 kPa to 100 kPa, a regression line formula (Y=aX) passing through the origin is obtained, and an additional 1 representing a 1% outer diameter change rate is added to the right side of the formula (Y=aX+1). A straight line based on the derived formula is added to the above graph, and the highest pressure among the pressures of the curve of the outer diameter change rate that does not exceed the straight line is set as the elastic limit pressure of the measurement component.

[0132] The test was performed on 6 or more measurement modules, and the average value thereof was made the elastic limit pressure of the porous hollow fiber membrane.

[0133] (2) Method for measuring inner diameter and film thickness

[0134] The cross-sectional slices of the porous hollow fiber membrane were prepared and photographed at 200 times using a microscope (Keyence Corporation, Model VHX-5000) to prepare images. The membrane thickness of the hollow fiber cross section on the image was measured at least 20 places around the whole circumference, and the average value was set as the measured value of the membrane thickness. For the inner diameter, the area of ​​the hollow part of the hollow fiber cross section of the same image was obtained by image processing, and calculated as the diameter when a circular approximation was performed.

[0135] (3) Method for measuring water permeability of porous hollow fiber membrane

[0136] Ten porous hollow fiber membranes were bundled, a polyethylene tube connectable to a water permeability measuring machine was attached to one end with an adhesive, and the other end of the hollow fiber was adjusted and sealed to have an effective length of 16 cm to prepare a measurement module.

[0137] The water permeability measuring device comprises a mechanism for spraying water at a constant pressure from a conduit portion of a polyethylene tube that can be connected to a measuring component; a mechanism for quantifying the amount of sprayed liquid with high precision; a mechanism for measuring the quantitative time of the sprayed liquid; a bathtub for immersing the measuring component; and a mechanism for regulating the temperature of the sprayed water and the bath water.

[0138] The measuring module was immersed in a 37°C water bath, and the polyethylene tube of the measuring module was connected to the conduit of the water permeation measuring machine, and the time for 1 mL of 37°C water to flow at 27 kPa was measured. The filtration membrane area calculated based on the result of measuring the inner diameter (μm) of the porous hollow fiber membrane prepared under the same conditions as the porous hollow fiber membrane of the measuring module and the measured value of the 1 mL water flow time were used to calculate the permeability per 1 m 2 Membrane area, water permeability in 1 hour (L / (m 2 ·hr).

[0139] The test was performed on three or more evaluation modules, and the average value thereof was made the water permeability of the porous hollow fiber membrane.

[0140] (4) Bubble point determination method of porous hollow fiber membrane

[0141] One end of the porous hollow fiber membrane was sealed, and the other end was fixed to a metal connector using polyurethane resin in a manner that could be pressurized with air or nitrogen to form a test assembly (effective length 8 cm). A tube was installed in the test assembly, and 3M Novec 7200 high-functional liquid (trademark, manufactured by 3M Japan Limited) was injected into the tube to immerse the porous hollow fiber membrane in the liquid.

[0142] The bubble point measuring device includes a pressure regulating mechanism capable of gradually increasing the pressure by pressurizing the inner surface side of the porous hollow fiber membrane through a metal coupling, and a pressure display mechanism, and includes a flow meter capable of measuring the flow rate of gas flowing out of the tube of the test module.

[0143] The end of the pressurizing mechanism is installed on the metal connector of the test assembly, and the pipeline of the flow measuring mechanism is installed on the end of the test assembly tube. The pressure (MPa) when the gas flow rate leaking during the slow pressure increase is 2.4mL / minute is detected. The test is performed on more than 3 test assemblies, and the average value is set as the bubble point value.

[0144] (5) Method for determining virus LRV of porous hollow fiber membranes

[0145] Using known techniques, 0.001 m 2 The membrane area is made of Japanese Patent Publication No. 2013-17990 Figure 1 A small membrane module described in.

[0146] The solution to be filtered is prepared by the method described in the following (5-A) or (5-B).

[0147] (5-A) Preparation of a protein solution containing a virus. First, a polyclonal antibody (human IgG) (Venoglobulin-IH (human immunoglobulin for intravenous injection), manufactured by Benesis Corporation) is used and diluted with water for injection (Otsuka Pharmaceutical) in a manner such that the antibody concentration is 1 mg / mL to obtain an antibody solution. In addition, the salt concentration is adjusted to 0.1 mol / L using a 1 mol / L NaCl aqueous solution. Furthermore, the hydrogen ion index (pH) is adjusted to 4.0 using 0.1 mol / L HCl or 0.1 mol / L NaOH to make it a protein solution. 1.0 vol% of porcine parvovirus (PPV, the Association of Biological Products for Animal Use) is added to the obtained protein solution and stirred thoroughly to obtain a protein solution containing the virus.

[0148] As a virus-containing solution (5-B), 0.2% porcine parvovirus (PPV, type VR742, purchased from the American Cell Culture Collection (hereinafter, ATCC)) was added to an aqueous solution of pH 4.5, 0.02 mol / L acetic acid, and 0.1 mol / L NaCl to prepare an aqueous solution.

[0149] Use the prepared 0.001m 2 The small membrane module and the above-mentioned protein solution containing the virus or the solution containing the virus are filtered by the dead-end internal pressure filtration method until the filtration volume reaches 150 L / m 2 When using the virus-containing solution (5-B), filter until the filtration volume reaches 5 L / m 2 The filtration pressure is selected according to the elastic limit pressure of the porous hollow fiber membrane. For porous hollow fibers with an elastic limit pressure of less than 200 kPa, 98 kPa is set as the adaptive pressure for implementation. For porous hollow fibers with an elastic limit pressure of more than 200 kPa, 196 kPa is set as the adaptive pressure for implementation.

[0150] Next, in order to measure the virus infection titer, Dulbecco's modified Eagle's medium (1X) containing 3% BenchMark bovine serum albumin (trademark, manufactured by Gemini Bio-Products) and 1% PENICILLINSTREPTOMYCIN SOL (trademark, manufactured by Life Technologies Corporation) was prepared after heating in a 56°C water bath for 30 minutes to inactivate the virus, and a solution of liquid + 4.5 g / L D-Glucose + L-Glutamine-Sodium Pyruvate (trademark, manufactured by Life Technologies Corporation, hereinafter referred to as D-MEM) (hereinafter referred to as 3% FBS / D-MEM) was prepared. The filtered stock solution and the filtrate were respectively diluted to 10 times and 10 times with 3% FBS / D-MEM. 2 times, 10 3 times, 10 4 Times and 10 5 times.

[0151] PK-13 cells (No.CRL-6489, purchased from ATCC) were diluted with 3% FBS / D-MEM to prepare a cell concentration of 2.0×10 5(cells / mL) diluted cell suspension was dispensed into all wells of 10 96-well round-bottom cell culture plates at 100 μL each. In addition, 100 μL each of the prepared filtered stock solution and its dilution, filtrate and its dilution were dispensed into every 8 wells. Then, cells were cultured for 10 days at 37°C and 5% carbon dioxide atmosphere.

[0152] The cells cultured for 10 days were subjected to 50% tissue culture infection dose (TCID) using the hemadsorption method (see General Guide to Virus Experiments, edited by the National Institute of Preventive Health Student Association, p. 173). 50 ) determination.

[0153] That is, the method is as follows: chicken preserved blood (trademark, manufactured by NIPPON BIOTEST LABO) is diluted 5 times with PBS (-) adjusted solution of Dulbecco PBS (-) powder (trademark, manufactured by Nissui Pharmaceutical Co., Ltd.), and then centrifuged at 2500 rpm, 4°C, and 5 minutes. The supernatant is removed by suction, and the obtained precipitate is diluted 200 times again with PBS (-) adjusted solution. The diluted solution is dispensed into all wells of a cell culture plate at 100 μL each, and after standing for 2 hours, the adsorption of red blood cells to the cell tissue surface is observed to evaluate virus infection. The virus infection ratio of the filtered stock solution, the filtrate, and each diluted solution is confirmed, and the infection titer (TCID) is calculated by the Spearman-Karber calculation formula. 50 / mL).

[0154] The logarithmic virus removal rate (LRV) is calculated by LRV = log 10 (C0 / C F ) is calculated, where C0 represents the infection titer of the filtered stock solution (TCID 50 / mL)、C F The TCID represents the infectious titer of the filtrate after filtering the filtrate with a virus removal membrane. 50 / mL).

[0155] For the use of 0.001m 2 The filtration rate of the protein solution containing the virus in the membrane module was measured until the filtration rate reached 150 L / m 2 The time until 1m 2 Membrane area, filtration volume per hour (L / (m 2 ·hr)) calculation.

[0156] (6) 0.001m 2 Method for determining water permeability of membrane modules

[0157] Prepare 0.001m 2The membrane module is filtered by internal pressure filtration and dead-end method at a temperature of 25°C and a pressure difference of 98 kPa between membranes for 10 minutes. The filtrate is measured as the filtrate per 1m 2 Membrane area, water permeability in 1 hour (L / (m 2 ·hr)) calculation.

[0158] (7) 0.001m 2 Determination of Colloidal Gold LRV of Membrane Modules

[0159] A solution of colloidal gold having a particle size of about 20 nm, AGP-HA20 (trademark, manufactured by Asahi Kasei Medical Co., Ltd.), was diluted with distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and a 0.27 mass % SDS (sodium lauryl sulfate) aqueous solution, and the solution was adjusted so that the absorbance at a wavelength of 526 nm measured by an ultraviolet / visible spectrophotometer (manufactured by Shimadzu Corporation, model UV-2450) became 1.00 to prepare a colloidal gold solution filtration stock solution.

[0160] Prepare 0.001m 2 The membrane assembly uses the prepared colloidal gold solution to filter the membrane by internal pressure filtration, dead end method at a temperature of 25°C, a membrane pressure difference of 25 kPa, and a filtration volume of 2 L / m 2 Filtration was carried out under the conditions of 0.5L / m 2 ~2.0L / m 2 The filtrate was sampled.

[0161] The absorbance of the filtered stock solution and the filtrate at a wavelength of 526 nm was measured using an ultraviolet / visible spectrophotometer (manufactured by Shimadzu Corporation, model UV-2450). 10 The logarithmic removal rate (LRV) of the colloidal gold particles was calculated using the formula (A / B), where A represents the absorbance of the filtered stock solution and B represents the absorbance of the filtrate.

[0162] [Measurement results of physical properties of porous hollow fiber membrane]

[0163] The elastic limit pressure, inner diameter (R), membrane thickness (t), water permeability and bubble point of a porous hollow fiber membrane virus filter containing regenerated cellulose (Planova S20N, registered trademark, Asahi Kasei) were measured by the above-mentioned various measurement methods, and the results are shown in Table 1. In addition, using this porous hollow fiber membrane virus filter, a membrane area of ​​0.001 m 2 The method of making the same as that of Japanese Patent Publication No. 2013-17990 Figure 1 Similar small membrane modules, as 0.001m 2Table 1 shows the measurement results of virus LRV (using the virus-containing protein solution described in (5-A) of the above measurement method example), water permeability, and colloidal gold LRV of the membrane module.

[0164] Furthermore, in order to confirm that the performance of the porous hollow fiber membrane does not change during the integrity test, the 0.001m 2 When the outer surface area of ​​the porous hollow fiber membrane of the membrane assembly is filled with pure water, the inner surface side of the porous hollow fiber membrane is pressurized with air at 250 kPa for 10 minutes, and then the water permeability and colloidal gold LRV are measured to obtain one result. Another measurement result is obtained without the aforementioned pressurization. The ratio of the water permeability and colloidal gold LRV before and after pressurization, which is used to represent the difference between the two results, is shown in Table 1.

[0165] Here, the inventors chose colloidal gold removal performance evaluation as an alternative method for virus removal performance evaluation because the virus evaluation method has a measurement limit depending on the virus concentration in its solution, and the virus solution contains a large number of non-infectious particles, etc. Therefore, it is preferred to evaluate the removal performance of colloidal gold particles for determining subtle differences in membrane structure.

[0166] [Table 1]

[0167] Elastic limit pressure(kPa) 360 Inner diameter (R, μm) 279.8 Film thickness (t, μm) 45 Ratio of inner diameter to film thickness (R / t) 6.2 <![CDATA[Water permeability (L / (m 2 ·hr))※1]]> 30.8 Bubble point(Mpa) 1.89 Parvovirus (PPV)LRV >5.5 <![CDATA[0.001m 2 Membrane module water permeability (L / (m 2 hr)〉※2]]> 58.9 <![CDATA[After pressurizing at 50 kPa for 10 minutes, 0.001 m 2 Water permeation rate of the membrane module (L / (m 2 ·hr)) ※2]]> 64.9 Change in water permeability caused by 250kPa pressure for 10 minutes (ratio of back / front) 1.11 <![CDATA[0.001m 2 Membrane Module Colloidal Gold LRV]]> 1.73 <![CDATA[After pressurizing at 50 kPa for 10 minutes, 0.001 m 2 Water permeation rate of the membrane module (L / (m 2 ·hr))]]> 1.71 Change in water permeability caused by 250kPa pressure for 10 minutes (ratio of back / front) 0.99

[0168] ※1 Filtration conditions: 27kPa, 37℃ ※2 Filtration conditions: 98kPa

[0169] The filtration rate of the virus-containing protein solution measured using a porous hollow fiber membrane-type virus filter (Planova S20N, registered trademark, Asahi Kasei) containing regenerated cellulose was 145 LMH.

[0170] [Example 1 and Comparative Example 1]

[0171] (1) Preparation of culture supernatant

[0172] The culture fluid containing CHO cells producing monoclonal antibody mAb A (pembrolizumab, genetically recombinant humanized IgG4 monoclonal antibody, molecular weight of about 149,000) was filtered with a filter membrane (manufactured by Asahi Kasei Medical Corporation, trade name BioOptimal (registered trademark) MF-SL) to obtain impurities and a culture supernatant containing the antibody. The culture supernatant contained 1.5 g / L of monoclonal antibody mAb A in terms of antibody protein.

[0173] (2) Preparation of antibody solution (purification of culture supernatant)

[0174] In a typical commercial method, the culture supernatant obtained in (1) is purified by protein A affinity chromatography and ion exchange chromatography, and the purified solution is buffer exchanged with a 15mmol / L acetic acid buffer (pH5.0, 22mS / cm) containing sodium chloride to prepare a solution containing antibody mAb A. In the pH determination of the buffer, a pH meter HM-30R (manufactured by DKK-TOA CORPORATION) is used. In addition, the conductivity of the buffer is measured using a conductivity meter CM-42X (manufactured by DKK-TOA CORPORATION). It should be noted that the pH and conductivity values ​​of the buffer described later are also obtained by the same measuring device. The prepared antibody solution is analyzed using a spectrophotometer NanoDrop One (manufactured by Thermo Fisher Scientific) to measure the antibody concentration, and the result is 15.4mg / mL.

[0175] (3) DLS measurement

[0176] The solution containing antibody mAb A prepared in (2) was poured into a polystyrene cuvette, and the particle size distribution of antibody mAb A contained in the solution was measured using a dynamic light scattering (DLS) measuring device Zetasizer nano ZSP (manufactured by Spectris Corporation) under the following conditions. Three measurements were performed for one sample, and the average value thereof was obtained. The obtained average value is shown in Figure 2 The maximum value of the particle size is 28.2 nm, and the particle size with the highest scattering intensity is 13.5 nm. In addition, the particle size distribution of antibody mAb A includes a range of 22.0 nm or more.

[0177] Dynamic light scattering method

[0178] · Sample: Protein

[0179] Dispersion medium: water

[0180] Temperature: 25°C, Equilibration time: 15 seconds

[0181] Sample cell: Polystyrene sample cell ZEN0040

[0182] Measurement angle: 173° backscatter (NIBS default value)

[0183] ·Measurement time: Automatic

[0184] ·Number of measurements: 3 times

[0185] Detailed settings: Automatic selection of attenuation → Yes

[0186] (4) HIC (Hydrophobic Interaction Chromatography) Determination

[0187] The solution containing the antibody mAb A prepared in (2) was diluted with pure water to an antibody concentration of 0.3 mg / mL, and the time from the start of antibody supply to the column of the apparatus to detection by the detector was measured using a high performance liquid chromatograph Prominence (Shimadzu Corporation) under the following conditions.

[0188] Guard column: Filter module NPR (manufactured by Tosoh Corporation) with a built-in membrane filter (Fluoropore 0.45 μm FP-045, manufactured by Tosoh Corporation)

[0189] Main column: TSKgel Butyl-NPR (particle size 2.5 μm, inner diameter 4.6 mm, length 10 cm, manufactured by Tosoh Corporation)

[0190] Column temperature: 35°C

[0191] Flow rate: 1.0mL / min

[0192] Injection volume: 100μL

[0193] Detection wavelength: 280nm

[0194] Buffer A: 20mmol / L phosphate buffer + 1.5mol / L ammonium sulfate (pH 7.0)

[0195] Buffer B: 20mmol / L phosphate buffer (pH7.0)

[0196] Gradient: The proportion of buffer B was gradually increased at a constant rate from 0% at the start of measurement and reached 100% after 25 minutes.

[0197] Among the detection peaks of retention time measured under the above conditions, the time of the highest detection peak is set as Tr, such as Figure 3 As shown, the Tr of antibody mAb A is 19.12 minutes.

[0198] The time when the antibody contained in the solution reaches the detector through the hydrophobic interaction chromatographic column is affected by the length of the piping of the hydrophobic interaction chromatographic device. In order to study this effect, the guard column and the main column are removed from the device, and a stainless steel joint for analysis (made by Shimadzu Corporation, product number 228-16004-13) is installed to analyze mAb A, and the result is a detection peak at 0.22 minutes. The time of the detection peak when measured in the absence of the column is set to Tm. The Tr-Tm of the antibody mAb A that eliminates the influence of the piping length is 18.90 minutes.

[0199] In addition, the time it takes for an antibody contained in a solution to pass through a hydrophobic interaction chromatography column and reach a detector is affected by the piping length of the hydrophobic interaction chromatography apparatus and the measurement conditions. Therefore, the following retention coefficient k value is defined as an indicator representing the hydrophobicity of the antibody that is not affected by these factors.

[0200] k=(Tr-T0) / (T0-Tm)

[0201] Tr: Detection time of the maximum peak (minutes)

[0202] T0: Peak detection time when the antibody is passed directly without interacting with the column (minutes)

[0203] Tm: Peak detection time when measured without a column (minutes)

[0204] In hydrophobic chromatography, the detection time of the peak can vary according to the salt concentration of buffer A, and T0 can be expressed by the detection time of the peak at the moment when the detection time of the peak remains unchanged even if the salt concentration of buffer A is changed from high concentration to low concentration. Buffer A of Example 1 (4) was set to 20mmol / L phosphate buffer + 3.0mol / L sodium chloride (pH7.0), and 20mmol / L phosphate buffer + 1.0mol / L sodium chloride (pH7.0) to analyze mAb B (nivolumab, genetically recombinant human IgG4 monoclonal antibody). The detection time was 0.96 minutes, so it was set to T0. The k value was calculated based on the above Tm, Tr, and T0. As shown in Table 4, the k value is 24.5.

[0205] Therefore, the Tr-Tm of antibody mAb A in hydrophobic interaction chromatography is 15 minutes or more and the k value is 19 or more.

[0206] (5) Preparation of virus removal filter of comparative example

[0207] A hydrophilized PVDF hollow fiber membrane was prepared by the same method as described in

[0061] and

[0062] of Japanese Patent No. 5755136, with an effective membrane area of ​​0.000075 m 2 The virus removal filter of the comparative example is sometimes referred to as VFA below.

[0208] (6) Measurement of Flux Decay in Comparative Example

[0209] The solution containing antibody mAb A prepared in (2) was passed through the virus removal filter of the comparative example prepared in (5) at a fixed pressure of 196 kPa at the dead end. The antibody solution treatment rate per unit area per unit time of the virus removal filter of the comparative example was measured (LMH = treatment liquid volume (L) / virus removal membrane area (m 2 ) / time), get Figure 4 The results are shown. In addition, the following flux attenuation (%) was calculated as an index of clogging of the virus removal filter.

[0210] Flux attenuation (%) = (V120-V5) / V5

[0211] V120: Antibody solution processing rate 120 minutes after the start of filtration (LMH)

[0212] V5: Antibody solution processing speed 5 minutes after filtration started (LMH)

[0213] V5 is 32. Since the antibody solution treatment rate (LMH) is already 3.2 55 minutes after the start of filtration, V120 is less than 3.2. Therefore, the flux attenuation of VF B is more than 90%. Therefore, even a virus removal filter capable of filtering under high pressure will be severely clogged when filtering a solution containing antibody mAb A, and the filtration rate will be greatly reduced.

[0214] (7) Preparation of virus removal filter of Example

[0215] A hollow fiber virus filter (Planova S20N, registered trademark, Asahi Chemical) containing regenerated cellulose was prepared with an effective membrane area of ​​0.000075 m 2 The virus removal filter of the example was prepared in the manner of . The virus removal filter of the example may be hereinafter referred to as VFB.

[0216] (8) Measurement of Flux Attenuation in Example

[0217] Using a solution containing antibody mAb A, the LMH of VF B was measured in the same manner as for V A A, and the result was Figure 4 The results shown. The flux attenuation of VF B is 28.6% or more. Therefore, VF B has a smaller flux attenuation when filtering mAb A than VF A, and exerts a stable filtering performance. Thus, VF B is an antibody with a particle size distribution of more than 22.0 nm, indicating that antibodies with a Tr-Tm of more than 15 minutes and a k value of more than 19 in hydrophobic interaction chromatography can be purified while suppressing clogging. It should be noted that the filtering conditions of the subsequent virus removal filter are shown in Table 2.

[0218] [Examples 2 to 4]

[0219] The solutions containing mAb A prepared by the same methods as in Example 1 (1) and (2) were buffer exchanged with a 15 mmol / L acetic acid buffer (pH 5.5, 20 mS / cm) containing sodium chloride by the same method as in Example 1 (2) to prepare a solution containing mAb A of Example 2 having an antibody concentration of 10.0 mg / mL, a solution containing mAb A of Example 3 having an antibody concentration of 30.0 mg / mL, and a solution containing mAb A of Example 4 having an antibody concentration of 50.0 mg / mL.

[0220] These solutions containing mAb A were filtered with VFB, and LMH was measured by the same method as in Example 1 (6). Figures 5 to 7 The results shown show that the flux attenuation was 0% when the antibody concentration was 10.0 mg / mL, 50.0% when the antibody concentration was 30.0 mg / mL, and 75.0% when the antibody concentration was 50.0 mg / mL. Generally, the higher the antibody concentration in the solution, the more likely it is that antibodies will associate with each other and the antibodies will be adsorbed to the membrane and cause clogging, but it is shown that even in such a solution with a high antibody concentration, VF B has less clogging than VF A.

[0221] [Examples 5 to 7]

[0222] For the solutions containing mAb A prepared by the same method as in Example 1 (1) and (2), the antibody concentration, pH, conductivity and buffer type were changed to the conditions shown in Table 2 to prepare solutions containing mAb A in Examples 5 to 7. The solutions containing mAb A in Examples 5 to 7 were filtered with VFB, and LMH was measured by the same method as in Example 1 (6). Figures 8 to 10 The results shown showed that the flux attenuation of the solution containing mAb A in Example 5 was 1.5%, the flux attenuation of the solution containing mAb A in Example 6 was 18.0%, and the flux attenuation of the solution containing mAb A in Example 7 was 83.3%. Generally, the higher the conductivity in the solution, the more likely it is that antibodies will associate with each other and clogging due to antibody adsorption to the membrane will occur, and the pH of the solution will also affect the filtration performance of the filter, but it is shown that even if the pH and conductivity change, VFB has less clogging than VFA.

[0223] [Example 8 and Comparative Example 2]

[0224] The same experiment as in Example 2 was performed by changing the antibody type of the antibody solution. A solution containing the monoclonal antibody bevacizumab (manufactured by Pfizer) was buffer exchanged with a 20 mmol / L acetate buffer (pH 5.5, 20 mS / cm) containing sodium chloride to prepare a bevacizumab solution having an antibody concentration of 10.0 mg / mL.

[0225] The particle size distribution of particles contained in the solution containing bevacizumab was measured by DLS in the same manner as in Example 1 (3). The results were as follows: Figure 2 As shown in FIG. 1 , the maximum particle size of the particles contained in the solution containing bevacizumab is 24.4 nm, and the particle size with the largest scattering intensity is 11.7 nm. In addition, the particle size distribution of bevacizumab includes a range of 22.0 nm or more. The retention time of bevacizumab based on HIC was measured by the same method as in Example 1 (4). The results are as follows: Figure 3 As shown in FIG. 1 , the retention time of the highest peak is 19.24 minutes. Therefore, the Tr-Tm of bevacizumab in the hydrophobic interaction chromatography is 15 minutes or more, and the k value is 19 or more.

[0226] As Comparative Example 2, a solution containing bevacizumab was filtered with VFA, and as Example 8, a solution containing bevacizumab was filtered with VFB. The LMH of Example 8 and Comparative Example 2 was measured by the same method as in Example 1 (6). As a result, Fig.11 The results are shown. The flux attenuation when using VFA in Comparative Example 2 was 61.4%, and the flux attenuation when using VFB in Example 8 was 12.3%. As in the case where the antibody was mAb A, in the case of bevacizumab, the blockage of VFB was also shown to be smaller than that of VFA.

[0227] [Example 9 and Comparative Examples 3 and 4]

[0228] The same experiment as in Example 2 was performed by changing the antibody type of the antibody solution. The solution containing the bispecific antibody, Bemelizumab (UCB Japan), was buffer exchanged with a 15 mmol / L acetate buffer (pH 5.5, 22 mS / cm) containing sodium chloride to prepare a solution containing Bemelizumab with an antibody concentration of 15.4 mg / mL.

[0229] The particle size distribution of particles contained in the solution containing Bimelizumab was measured by DLS in the same manner as in Example 1 (3). The results were as follows: Figure 2As shown in FIG. 1 , the maximum particle size of the particles contained in the solution containing Bemelfit monoclonal antibody is 24.4 nm, and the particle size with the largest scattering intensity is 13.5 nm. In addition, the particle size distribution of Bemelfit monoclonal antibody includes a range of 22.0 nm or more. The retention time of Bemelfit monoclonal antibody based on HIC was measured by the same method as in Example 1 (4). The results are as follows: Figure 3 As shown in the figure, the retention time of the highest peak is 16.16 minutes. Therefore, the Tr-Tm of Bimelikimab in the hydrophobic interaction chromatography is more than 15 minutes, and the k value is more than 19.

[0230] As Comparative Example 3, the solution containing Bemelech was filtered with VFA. In addition, as Example 9, the solution containing Bemelech was filtered with VFB. The LMH of Example 9 and Comparative Example 3 was measured by the same method as in Example 1 (6), and the results were as follows: Fig.12 The results are shown. The flux attenuation when using VFA in Comparative Example 3 was 51.1%, and the flux attenuation when using VFB in Example 9 was 12.7%. As in the case where the antibodies were mAb A and bevacizumab, in the case of bevacizumab, the blockage of VFB was smaller than that of VFA.

[0231] In addition, a PES filter VFC was prepared by the method described in Example 1 of International Publication No. 2020 / 203716. As Comparative Example 4, a solution containing Bemelech monoclonal antibody was filtered with VFC, and the LMH of Comparative Example 4 was measured by the same method as in Example 1 (6). The result was Fig.13 The results are shown in Figure 2. The flux attenuation when using VFC in Comparative Example 4 was 69.1%. Therefore, it is shown that the clogging of VFB is smaller than that of VFC.

[0232] [Comparative Example 5]

[0233] As an antibody protein, a culture supernatant containing 2.3 g / L of the monoclonal antibody mAb B (nivolumab, a recombinant human IgG4 monoclonal antibody, with a molecular weight of approximately 145,000) was prepared, and an antibody solution was prepared by the same method as (1) and (2) of Example 1, thereby preparing a solution containing mAb B having a liquid property of 15 mmol / L acetic acid buffer (pH 5.5, 15 mS / cm) containing sodium chloride and an antibody concentration of 14.6 mg / mL.

[0234] The particle size distribution of particles contained in the solution containing mAb B was measured by DLS in the same manner as in Example 1 (3). The results were as follows: Fig.14As shown in FIG. 1 , the maximum particle size of the particles contained in the solution containing mAb B is 21.0 nm, and the particle size with the largest scattering intensity is 11.7 nm. In addition, the particle size distribution of mAb B does not include a range of 22.0 nm or more. The detection time of mAb B based on HIC was measured by the same method as in Example 1 (4). The results are as follows: Figure 3 As shown in the figure, the detection time of the highest peak is 14.46 minutes. Therefore, the Tr-Tm of antibody mAb B in hydrophobic interaction chromatography is less than 15 minutes and the k value is less than 19.

[0235] As Comparative Example 5, the solution containing mAb B was filtered with VFA, and the LMH of Comparative Example 5 was measured by the same method as in Example 1 (6). Fig.15 The flux attenuation of the solution containing mAb B in Comparative Example 5 was 19.4%, and no clogging occurred to the same extent as mAb A in Comparative Example 1, bevacizumab in Comparative Example 2, and bevacizumab in Comparative Example 3. Therefore, it is shown that antibodies that do not contain an antibody with a particle size distribution of 22.0 nm or more or antibodies with a Tr-Tm of less than 15 minutes and a k value of less than 19 in hydrophobic interaction chromatography are not likely to cause clogging even in the case of VFA.

[0236] The results of DLS measurements of the antibodies of Comparative Examples 1 to 4, which were significantly blocked when using VF A, and the antibodies of Comparative Example 5, which were less blocked, are shown in Table 3. The maximum particle size in the particle size distribution of the antibodies of Comparative Examples 1 to 4 was 24.4 nm or more, while the maximum particle size in the particle size distribution of the antibody of Comparative Example 5 was 21.0 nm. In the case of DLS, the association between particles is reflected in the measured particle size. The results shown in Table 3 show that the antibodies that were significantly blocked when using VF A are antibodies that are prone to association between antibodies. Generally, multispecific antibodies are asymmetric between antigen binding sites in terms of charge and hydrophobicity, and it is believed that antibodies are prone to association with each other compared to monoclonal antibodies. However, in the case of VF B, even if it is a solution containing antibodies that are prone to association, the flux attenuation is low, thus showing that VF B is suitable for filtration of solutions containing antibodies that are prone to association.

[0237] In addition, the comparison results of the Tr-Tm values ​​and k values ​​of mAb A, bevacizumab, bevacizumab, and mAb B are shown in Table 4. The Tr-Tm value of mAb B, which was less blocked when using VFA, was 14.24, while the Tr-Tm values ​​of mAb A, bevacizumab, and bevacizumab, which were significantly blocked when using VFA, were 18.90, 19.02, and 16.14, respectively, which were larger than the Tr-Tm value of mAb B. The k value of mAb B, which was less blocked when using VFA, was 18.3, while the k values ​​of mAb A, bevacizumab, and bevacizumab, which were significantly blocked when using VFA, were 24.5, 24.6, and 20.6, respectively, which were larger than the k value of mAb B. Therefore, it was shown that mAb A, bevacizumab, and bevacizumab were more hydrophobic than mAb B. Generally, antibodies with high hydrophobicity are easily adsorbed to filters and easily cause clogging. However, VFB is made of highly hydrophilic cellulose and has a high elastic limit pressure of 200 kPa or more. Therefore, it is shown that it is not easily affected by the hydrophobicity of antibodies and is not prone to clogging.

[0238] [Table 2-1]

[0239]

[0240] [Table 2-2]

[0241]

[0242] [Table 3]

[0243]

[0244] [Table 4]

[0245]

Claims

1. A method for purifying an antibody, comprising the following steps: preparing a solution comprising an antibody; and Purifying the antibody by filtering the solution using a porous membrane comprising regenerated cellulose, In the solution containing the antibody, the particle size distribution of the antibody includes a range of 22.0 nm or more.

2. The method for purifying an antibody according to claim 1, wherein: The porous membrane is a porous hollow fiber membrane.

3. The method for purifying an antibody according to claim 1, wherein: The elastic limit pressure of the porous membrane is 200 kPa or more.

4. The method for purifying an antibody according to claim 1, wherein: In the solution containing the antibody, the particle size distribution of the antibody includes a range of 24.0 nm or more.

5. The method for purifying an antibody according to claim 1, wherein: The antibody includes aggregates of the antibody.

6. The method for purifying an antibody according to claim 1, wherein: The antibody is an antibody capable of binding to two or more different antigens.

7. The method for purifying an antibody according to claim 1, wherein: The antibody is a monoclonal antibody.

8. The method for purifying an antibody according to claim 1, wherein: The antibody is contained in an antibody-drug conjugate.

9. The method for purifying an antibody according to claim 2, wherein: The ratio (R / t) of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t) is 8.4 or less.

10. The method for purifying an antibody according to claim 2, wherein: The membrane thickness (t) of the porous hollow fiber membrane is in the range of 20 μm or more and 70 μm or less.

11. The method for purifying an antibody according to claim 1, wherein: The regenerated cellulose is regenerated cellulose obtained by using a cuprammonia method.

12. The method for purifying an antibody according to claim 2, wherein: The pore size of the inner surface of the porous hollow fiber membrane is larger than the pore size of the outer surface.

13. The method for purifying an antibody according to claim 2, wherein: The porous hollow fiber membrane has an inclined structure in which the pore diameter decreases from the inner surface side toward the outer surface side.

14. The method for purifying an antibody according to claim 2, wherein: The water permeability of the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37°C is 10 L / (m 2 ·hr) and above and 50L / (m 2 hr) or less.

15. The method for purifying an antibody according to claim 2, wherein: The bubble point of the porous hollow fiber membrane is above 1.2 MPa.

16. The method for purifying an antibody according to claim 1, wherein: Viruses are removed during purification of the antibodies.

17. The method for purifying an antibody according to claim 16, wherein: The porous membrane has a parvovirus removal rate (LRV) of 4.0 or more.

18. The method for purifying an antibody according to claim 1, wherein: The pH of the solution containing the antibody is 4.5 or more and 10.0 or less.

19. The method for purifying an antibody according to claim 1, wherein: The salt concentration of the solution containing the antibody is 0 mmol / L or more and 1000 mmol / L or less.

20. The method for purifying an antibody according to claim 1, wherein: The conductivity of the solution containing the antibody is 0 mS / cm or more and 100 mS / cm or less.

21. A method for purifying an antibody, comprising the following steps: preparing a solution comprising an antibody; and Purifying the antibody by filtering the solution using a porous membrane comprising regenerated cellulose, (A) the Tr-Tm of the antibody in hydrophobic interaction chromatography is 15 minutes or more and / or (B) the k value of the antibody in hydrophobic interaction chromatography is 19 or more.

22. The method for purifying an antibody according to claim 21, wherein: The porous membrane is a porous hollow fiber membrane.

23. The method for purifying an antibody according to claim 21, wherein: In the porous membrane, the elastic limit pressure is 200 kPa or more.

24. The method for purifying an antibody according to claim 21, wherein: The Tr-Tm is 16 minutes or more and / or the k value is 20 or more.

25. The method for purifying an antibody according to claim 21, wherein: The antibody is an antibody capable of binding to two or more different antigens.

26. The method for purifying an antibody according to claim 21, wherein: The antibody is a monoclonal antibody.

27. The method for purifying an antibody according to claim 21, wherein: The antibody is contained in an antibody-drug conjugate.

28. The method for purifying an antibody according to claim 22, wherein: The ratio (R / t) of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t) is 8.4 or less.

29. The method for purifying an antibody according to claim 22, wherein: The membrane thickness (t) of the porous hollow fiber membrane is in the range of 20 μm or more and 70 μm or less.

30. The method for purifying an antibody according to claim 21, wherein: The regenerated cellulose is regenerated cellulose obtained by using a cuprammonia method.

31. The method for purifying an antibody according to claim 22, wherein: The pore size of the inner surface of the porous hollow fiber membrane is larger than the pore size of the outer surface.

32. The method for purifying an antibody according to claim 22, wherein: The porous hollow fiber membrane has an inclined structure in which the pore diameter decreases from the inner surface side toward the outer surface side.

33. The method for purifying an antibody according to claim 22, wherein: The water permeability of the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37°C is 10 L / (m 2 ·hr) and above and 50L / (m 2 hr) or less.

34. The method for purifying an antibody according to claim 22, wherein: The bubble point of the porous hollow fiber membrane is above 1.2 MPa.

35. The method for purifying an antibody according to claim 21, wherein: Viruses are removed during purification of the antibodies.

36. The method for purifying an antibody according to claim 35, wherein: The porous membrane has a parvovirus removal rate (LRV) of 4.0 or more.

37. The method for purifying an antibody according to claim 21, wherein: The pH of the solution containing the antibody is 4.5 or more and 10.0 or less.

38. The method for purifying an antibody according to claim 21, wherein: The salt concentration of the solution containing the antibody is 0 mmol / L or more and 1000 mmol / L or less.

39. The method for purifying an antibody according to claim 21, wherein: The conductivity of the solution containing the antibody is greater than or equal to 0 mS / cm and less than or equal to 100 mS / cm.

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