Porous adsorbent and separation column for biopharmaceutical purification using the same, and method for producing biopharmaceutical

Abstract

Disclosed is a porous adsorbent material for adsorbing and removing foreign substances from a biological drug material solution, the porous adsorbent material having a layer containing a nonionic polymer on at least one surface.

Description

TECHNICAL FIELD

[0001] The present application relates to a porous adsorbing material and a separation column for biopharmaceutical refinement using the same, and a method for manufacturing a biopharmaceutical. BACKGROUND

[0002] Porous materials typified by activated carbon and silica gel are materials having pores of a fine size, and have a property of easily adsorbing various substances. They are used for deodorants, drying agents, and in industry for separation of substances and the like, by making use of their characteristics.

[0003] Proteins are separated in various fields such as removal of pathogenic substances, environmental pollutants, and the like, in the manufacturing process of chemicals, foods, pharmaceuticals, cosmetics, and the like. As a method for separating proteins, widely known are mechanical or physical methods such as centrifugal separation, sedimentation, and the like; methods using separation materials such as solvent extraction, adsorption, chromatography, and the like.

[0004] In particular, in the process of manufacturing biopharmaceuticals such as antibody pharmaceuticals using biotechnologies such as cell culture technology, affinity chromatography using a protein A column, ion exchange chromatography, deep filtration, and the like are used as various protein separation technologies.

[0005] A protein adsorbing material used for separating proteins is required to have high adsorbing capacity, not to easily undergo clogging of meshes or deterioration due to adsorption of proteins and the like to the material used for separation, and to be able to exhibit stable performance for a long period of time, and the like.

[0006] Depending on the process, the concentration of antibodies in the process of manufacturing biopharmaceuticals is 2 to 3 times higher than that in blood. Therefore, in the case where a protein adsorbing material is used in the process of manufacturing biopharmaceuticals, not only high adsorbing capacity is required, but also clogging of meshes or deterioration is required to be less likely to occur compared to the case where the material adsorbs substances in blood.

[0007] As a method for increasing the adsorbing capacity of a protein adsorbing material, there are a method of increasing the adsorbing capacity, a method of chemically modifying the surface of the adsorbing material to increase the adsorbing selectivity, and the like. As a protein adsorbing material, a technology of using a porous solid fiber having an irregularly shaped cross section is disclosed (Patent Document 1). A method is described in which by making the fiber into an irregularly shaped cross section of a specific shape, an adsorbing material having an increased surface area per unit volume and high adsorbing performance is provided.

[0008] A protein adsorbing material easily adsorbs proteins that are not the object of adsorption on the surface of the material due to its high adsorbing property. In the case where a protein adsorbing material has fine pores, proteins adsorbed on the surface of the material enter the fine pores and change the size of the fine pores or hinder the entry of the fine pores, and thus there is a concern that the adsorption of the target proteins is hindered and the adsorbing performance is reduced.

[0009] As a method of inhibiting clogging of the mesh caused by adsorption of a protein to the surface of a material and maintaining high protein adsorption performance, there is a method of supporting a hydrophilic polymer on the surface of a material. For example, Patent Literature 2 describes a method of providing a protein adsorption material that inhibits a decrease in the ability to adsorb a protein and has high blood compatibility, by fixing a nitrogen-containing polymer containing a carboxylic acid vinyl ester to a surface.

[0010] Patent Literature 3 describes a method of providing a porous hollow fiber membrane that can both inhibit clogging of the mesh of a protein and separate a protein such as an antibody and a virus or the like in a short time and at a low pressure, by using a polysulfone-based polymer as a main component and supporting a polymer containing a monocarboxylic acid vinyl ester unit on a surface.

[0011] Patent Literature 4 describes a method of providing an adsorption bead that prevents the pores of the bead from being excessively hydrophilized by the hydrophilicity of a polymer, hindering the adsorption of a hydrophobic protein, or prevents the pores from being clogged by the polymer, reducing the adsorption performance, can maintain the adsorption performance of the porous bead, and improve blood compatibility, by supporting a polymer containing a monomer having a charge and a monomer having a specific structure as monomer units on the surface of a porous bead.

[0012] Patent Literature 5 describes a method of providing a blood cell removal adsorption body and a method of manufacturing the same, which can adjust the balance between hydrophilicity and hydrophobicity, reduce pressure loss in a blood cell adsorber, prevent blood from coagulating in the adsorber, and adsorb and remove a blood cell component, by fixing a highly hydrophilic polymer resin to the surface pores of a porous adsorption base material in a specific amount using an anchoring effect.

[0013] Prior Art Documents

[0014] Patent Literature

[0015] Patent Literature 1: International Publication No. 2016 / 067967

[0016] Patent Literature 2: Japanese Patent Application Publication No. 2014-207989

[0017] Patent Literature 3: International Publication No. 2019 / 225730

[0018] Patent Literature 4: Japanese Patent Application Publication No. 2020-6155

[0019] Patent Literature 5: Japanese Patent No. 5420341 SUMMARY

[0020] PROBLEMS TO BE SOLVED BY THE INVENTION

[0021] However, in Patent Document 1, the porous fiber surface was not treated with a hydrophilic polymer or the like. Proteins that are not the target of adsorption adhered to the fiber surface, which hindered the target protein from approaching the pores. There is a concern that the amount of target protein adsorbed may be reduced.

[0022] In patent documents 2-5, a hydrophilic polymer is loaded onto the surface of the protein adsorbent material to inhibit the adhesion of proteins to the surface of the protein adsorbent material. However, these documents only specify the amount of hydrophilic polymer used to control the adsorption and non-adsorption of proteins on the material surface, without describing the thickness of the hydrophilic polymer in the depth direction of the material.

[0023] If the hydrophilic polymer in the protein adsorbent material is thick in the depth direction, it may penetrate into the pores on the surface, changing the pore size, or it may inhibit protein adhesion, thus hindering the adsorption of the target protein and reducing the amount of protein adsorbed into the pores. Conversely, if the hydrophilic polymer is too thin in the depth direction, proteins that are not the target protein may adhere to the material surface, hindering the approach of the target protein to the pores and reducing the amount of protein adsorbed into the pores.

[0024] Through repeated research, the inventors discovered that by controlling the thickness of the material layer containing a nonionic polymer with hydrophilic units in the depth direction to a specific range, it is possible to obtain a protein adsorption material with high separation selectivity, where mesh blockage caused by the adsorption of proteins that are not adsorbed onto the material surface is suppressed.

[0025] The purpose of this invention is to provide a porous adsorbent material with high separation selectivity that can suppress mesh clogging caused by the adsorption of proteins that are not the target proteins and adsorb the target proteins, said porous adsorbent material being used to adsorb and remove impurities from biopharmaceutical raw material solutions.

[0026] Solution for solving the problem

[0027] To address the aforementioned issues, this invention has undergone repeated and in-depth research, resulting in the discovery that by incorporating a layer containing a nonionic polymer, a porous adsorbent material can be obtained that suppresses mesh clogging caused by the adsorption of proteins (which are not the target adsorbents) onto the material surface, and exhibits high separation selectivity. This porous adsorbent material is used to adsorb and remove impurities from biopharmaceutical raw material solutions. In other words, this invention is as follows.

[0028] A porous adsorbent material for adsorbing and removing impurities from a solution of biopharmaceutical raw materials, wherein at least one surface of the porous adsorbent material has a layer comprising a nonionic polymer.

[0029] Invention Effects

[0030] The porous adsorbent material according to the present invention can suppress mesh clogging caused by the adsorption of proteins that are not the target proteins and adsorb the target proteins. Therefore, it is suitable for adsorbing and removing impurities from biopharmaceutical raw material solutions. Detailed Implementation

[0031] The porous adsorbent material of the present invention is a porous adsorbent material having a layer comprising a nonionic polymer on at least one surface.

[0032] Furthermore, the present invention preferably comprises a protein adsorbent material with fine pores, having at least one surface having a layer containing a polymer that inhibits the adhesion of biological components, wherein the thickness of the layer containing the polymer that inhibits the adhesion of biological components is less than 3 μm when the cross-section of the aforementioned protein adsorbent material is analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS).

[0033] Here, porous adsorbent materials generally refer to materials with micropores that readily adsorb various substances. The substances adsorbed by such porous adsorbent materials are, for example, substances considered impurities in the raw material solutions of biopharmaceuticals; specific examples include substances described later, other proteins, lipids, and water.

[0034] Protein adsorbent materials refer to materials that possess the ability to adsorb proteins. Furthermore, the materials constituting porous adsorbent materials or protein adsorbent materials are not particularly limited, and examples include cellulose polymers such as polymethyl methacrylate (PMMA), polystyrene, polyethylene, polypropylene, cellulose acetate, cellulose diacetate, and cellulose triacetate; fluoropolymers such as polycarbonate, polyurethane, polyvinyl chloride, and polyvinylidene fluoride; polyesters such as polyacrylonitrile and polyethylene terephthalate; polyamides; silica used in quartz and silica gel; aluminosilicates used in zeolites; carbon fibers; and amorphous carbon used in activated carbon. Alternatively, copolymers or composites of these materials may also be used. Among these, a protein adsorbent material composed of a substrate containing at least one substance selected from PMMA, polystyrene, polypropylene, silica, aluminosilicate, and amorphous carbon exhibits excellent adsorption performance and is therefore preferred. A substrate containing at least one substance selected from PMMA, polystyrene, and polypropylene efficiently adsorbs the target protein; therefore, it is more preferred as a substrate constituting the protein adsorbent material. In particular, in PMMA, by mixing isotactic and syndiotactic components to form a stereocomposite as a three-dimensional composite, a substrate with uniform pore size can be obtained, which is therefore preferred. In particular, regarding polystyrene and polypropylene, it can be a core-sheath composite fiber with polystyrene as the core component and polystyrene and polypropylene as the sheath component.

[0035] Examples of shapes that can constitute porous adsorbent materials or protein adsorbent materials include fibers, nonwoven fabrics, woven fabrics, knitted fabrics, granules, membranes, and molded articles. Among these, fibers can increase their specific surface area by reducing their fiber diameter; therefore, the shape of the porous adsorbent material or protein adsorbent material of the present invention is preferably that of a fiber. It should be noted that, in the present invention, a fiber refers to either a solid fiber without continuous cavities in the length direction or a hollow fiber with cavities.

[0036] In hollow fibers, when only the inner side is in contact with a solution containing both the target protein and non-target proteins, the specific surface area is sometimes too small to effectively adsorb the target protein. Furthermore, even when the specific surface area is increased by contacting both the inner and outer sides of the hollow fiber with the solution containing both the target and non-target proteins, the flow rate of the solution on the inner side is slower than on the outer side of the hollow fiber, resulting in ineffective contact between the inner side and the solution, and sometimes insufficient removal of the target protein. Therefore, for obtaining high protein adsorption performance, solid fibers are preferred as the material constituting the protein adsorption material. Solid fibers can contain a single component or two or more components, and can have any shape such as an asymmetric structure, a homogeneous structure, an island structure, or a core-sheath structure. From the viewpoint of increasing protein adsorption capacity, a homogeneous structure with fine pores extending continuously into the interior of the solid fiber is preferred.

[0037] Furthermore, when the porous adsorbent or protein adsorbent is in the form of solid fibers or hollow fibers, the shape of the fiber cross-section is not particularly limited; it can be circular or any other shape. Preferably, it is made into a fiber with an irregular cross-sectional shape having protrusions, called fins, present on the outer periphery of the fiber cross-section. When the porous adsorbent or protein adsorbent is in the form of fibers, by having fins, an irregular shape is achieved, increasing the surface area per unit volume. As a result, an improvement in adsorption performance can be expected.

[0038] The number of tabs is not particularly limited, but as an upper limit, it is preferably 12 or less, more preferably 8 or less, and particularly preferably 6 or less. If the number of tabs is increased excessively, the gap between the tabs may become narrow, the surface area per unit volume may decrease, or the liquid being treated may have difficulty contacting the tabs.

[0039] When using fibers with irregular cross-sectional shapes as porous adsorbents or protein adsorbents, the protrusions of adjacent fibers sometimes interlock, hindering the contact between the fiber surface and the solution. Therefore, when using fibers with irregular cross-sectional shapes as protein adsorbents, it is preferable to prepare them as a blend with circular or elliptical fibers that do not have protrusions.

[0040] The porous adsorption material of this invention is used to adsorb and remove impurities from biopharmaceutical raw material solutions. Biopharmaceuticals refer to drugs manufactured using biotechnology such as gene recombination and cell culture, with proteins such as antibodies, hormones, and enzymes as active ingredients. The active ingredient of a biopharmaceutical is produced by cells, and a solution containing the active ingredient is isolated from the cells. The biopharmaceutical raw material solution refers to this isolated solution. Impurities refer to substances other than the active ingredient contained in the biopharmaceutical raw material solution. Examples of impurities include host cell protein (HCP), antibody degradation products, components derived from the culture medium, DNA, viruses, and free protein A.

[0041] The proteins targeted for adsorption as protein adsorbents are not particularly limited, and examples include albumin, globulin, and fibrinogen. For processes using biotechnology such as cell culture to manufacture biopharmaceuticals such as antibody drugs, various protein separation techniques are employed, including affinity chromatography such as protein A columns, ion exchange chromatography, and depth filtration. When the protein adsorbent of this invention is used in the manufacturing process of biopharmaceuticals, examples of proteins targeted for adsorption include host cell protein (HCP), antibody degradation products, components derived from the culture medium, DNA, viral particles, and free protein A.

[0042] The porous adsorbent material of the present invention has a layer comprising a nonionic polymer on at least one surface. A nonionic polymer is a polymer that is electrically neutral as a whole. Electroneutrality means that the pH in solution is in the range of 6.0 to 7.5. If it falls within this range, it can be a polymer containing zwitterions. In addition to water, the solvent used to prepare the solution can also be water containing organic solvents such as ethanol.

[0043] Among nonionic polymers, hydrophilic polymers are more preferred. Hydrophilic polymers are defined as polymers having hydrophilic functional groups such as ether groups, ester groups, amide groups, hydroxyl groups, and carboxyl groups in their polymer structure. These functional groups preferably account for more than 9% by weight of the total polymer weight.

[0044] Among these nonionic and hydrophilic polymers, those that inhibit the adhesion of biological components are more preferred. Polymers that inhibit the adhesion of biological components refer to those used to create flat films and which, when in contact with human blood, exhibit a viscosity of 4.3 × 10⁻⁶.3 μm 2 The polymer exhibits a platelet attachment count of 20 or less per area. There are no particular limitations on the method for fabricating a flat film using this polymer. For example, it can be fabricated by impregnating a polysulfone substrate with a solution of the polymer being examined, and then immobilizing the polymer by crosslinking it to the polysulfone substrate through gamma irradiation.

[0045] The specific steps for determining platelet attachment count are explained below. Regarding platelet attachment count, immediately after collecting human venous blood, heparin is added at a concentration of 50 U / ml. Within 10 minutes of blood collection, the platelet is brought into contact with the aforementioned flat membrane and shaken at 37°C for 4 hours. Subsequently, the flat membrane is washed with physiological saline, and blood components are fixed using glutaraldehyde-saline solution, followed by washing with distilled water. The flat membrane is then dried under reduced pressure at 0.5 Torr at room temperature for 10 hours. A Pt-Pd film is formed on the surface of the flat membrane by sputtering, serving as the sample. The surface of the flat membrane is observed using a scanning electron microscope at 1500x magnification, and the platelet count is counted at every 4.3 × 10⁻⁶. 3 μm 2 The number of attached platelets. The average number of attached platelets from 10 different fields of view was taken as the platelet attachment count (platelets / 4.3×10). 3 μm 2 To achieve sufficient inhibition of biological component adhesion, the platelet adhesion count was 20 / 4.3 × 10⁻⁶. 3 μm 2 The preferred size is 10 pieces / 4.3 × 10. 3 μm 2 the following.

[0046] The layer containing the polymer that inhibits the adhesion of biological components is particularly preferably located on the surface that initially contacts the biological components when separating them using the porous adsorbent or protein adsorbent of the present invention. Here, biological components refer to substances that constitute an organism, specifically proteins, lipids, carbohydrates, etc.

[0047] There are no particular limitations on the method of contacting porous adsorbents or protein adsorbents with biological components, as long as the porous adsorbents or protein adsorbents can come into contact with biological components. Examples include: passing a solution containing biological components through a porous adsorbent or protein adsorbent, or immersing a solution containing biological components in a porous adsorbent or protein adsorbent.

[0048] If the layer containing the nonionic polymer or the polymer that inhibits the adhesion of biological components in a porous adsorbent material or protein adsorbent material is too thick, the polymer that inhibits the adhesion of biological components may sometimes enter the pores on the surface, causing changes in the pore size, or it may hinder adsorption by inhibiting the adhesion of biological components, thus reducing the amount of protein adsorbed into the pores. Therefore, the thickness of the layer containing the nonionic polymer or the polymer that inhibits the adhesion of biological components is preferably 5 μm or less, more preferably 3 μm or less. On the other hand, if the thickness of the layer containing the nonionic polymer or the polymer that inhibits the adhesion of biological components is too thin, proteins that are not the target of adsorption may sometimes adhere to the material surface, hindering the approach of the target proteins to the pores, thus reducing the amount of protein adsorbed into the pores. Therefore, the thickness of the layer containing the nonionic polymer or the polymer that inhibits the adhesion of biological components is preferably 1.5 μm or more, more preferably 2 μm or more. Here, the thickness of the layer containing the nonionic polymer or the polymer that inhibits the adhesion of biological components refers to the value obtained by performing compositional analysis on the cross-section of the protein adsorbent material using time-of-flight secondary ion mass spectrometry (TOF-SIMS). It should be noted that, due to the limitations of TOF-SIMS spatial resolution, the lower limit for thickness determination derived from the spectral profile of the cross-section is 0.2 μm. Thicknesses down to 10 nm can be determined by combining sample preparation using a surface / interface property analysis device (SAICAS (trademark)) and the tilting cutting method with TOF-SIMS deep profile determination using argon cluster ion beam (Ar-GCIB); or by using XPS. The thickness of the layer containing the polymer that inhibits the adhesion of biological components in the protein adsorbent material of this invention is not particularly limited if it is 3 μm or less.

[0049] Regarding the method for determining the thickness of a layer containing a nonionic polymer or a polymer that inhibits the adhesion of biological components by performing compositional analysis using time-of-flight secondary ion mass spectrometry (TOF-SIMS), more specifically, by detecting peaks originating from the aforementioned polymer that inhibits the adhesion of biological components through compositional analysis based on a TOF-SIMS device, information related to the amount of nonionic polymer or polymer that inhibits the adhesion of biological components can be obtained by analyzing its mass (m / z), and the thickness of the layer containing the nonionic polymer or polymer that inhibits the adhesion of biological components can be determined. As a method for determining the thickness, the methods described in the Examples section (Layer Thickness Measurement 1 and Layer Thickness Measurement 2) can be used.

[0050] In TOF-SIMS-based compositional analysis, pulsed ions (primary ions) are irradiated onto the surface of a sample placed in an ultra-high vacuum. Ions released from the sample surface (secondary ions) gain kinetic energy and are guided to a time-of-flight mass spectrometer. Secondary ions, accelerated with the same energy, pass through the analyzer at velocities corresponding to their mass, but the distance to the detector is fixed. Therefore, the time to reach the detector (time of flight) becomes a function of mass. By precisely measuring the distribution of this time of flight, the mass distribution of the secondary ions, i.e., the mass spectrum, can be obtained. For example, when using Bi3... ++ When the primary ion is detected and secondary negative ions are also detected, the peak at m / z = 26.01 corresponds to CN. - The peak at m / z = 42.01 corresponds to CNO. - It should be noted that, in cases where sufficient determination cannot be achieved using the aforementioned ionic species, the ionic species can be replaced with characteristic species from the nonionic polymer used or polymers that inhibit the adhesion of biological components.

[0051] The compositional analysis conditions based on TOF-SIMS determination are as follows. The measurement region was set to 50 μm × 50 μm, the primary ion acceleration voltage was set to 25 kV, and the pulse width was set to 125.0 ns. The detection depth in this method is below several nm. More specifically, the determination was performed according to "(1) TOF-SIMS determination" described later.

[0052] When a nonionic polymer or a polymer that inhibits the adhesion of biological components is present on at least one surface of a porous adsorbent material or a protein adsorbent material, the amount of the nonionic polymer or polymer that inhibits the adhesion of biological components on the surface of the porous adsorbent material or protein adsorbent material can be quantified by X-ray electron spectrometry (XPS). Here, the amount of the nonionic polymer or polymer that inhibits the adhesion of biological components on the surface of the porous adsorbent material or protein adsorbent material can be indicated by the following ratio: the ratio of nitrogen atoms to all atoms (atomic percentage) detected by XPS when measured from the layer side containing the nonionic polymer or polymer that inhibits the adhesion of biological components. If the ratio of nitrogen atoms to all atoms (atomic percentage) when measured by XPS from the layer side containing the nonionic polymer or polymer that inhibits the adhesion of biological components in the porous adsorbent material or protein adsorbent material is too high, it may sometimes hinder the approach of the protein, the target of adsorption, to the pores of the porous adsorbent material or protein adsorbent material, resulting in a reduction in the amount of protein adsorbed into the pores. On the other hand, if the proportion of nitrogen atoms relative to 100 (atomic percentage) of all atoms when measured using XPS is too low, proteins that are not the target of adsorption may adhere to the material surface, hindering the approach of the target proteins to the pores, and reducing the amount of protein adsorbed into the pores. Therefore, when measuring from the layer side containing nonionic polymers or polymers that inhibit the adhesion of biological components, the proportion of nitrogen atoms relative to 100 (atomic percentage) of all atoms detected by XPS is preferably 0.5 (atomic percentage) or more and 8 (atomic percentage) or less.

[0053] When measuring the proportion of nitrogen atoms detected by XPS from the side of a layer containing nonionic polymers or polymers that inhibit the adhesion of biological components, the XPS measurement angle used is the value measured at 45°. When measuring at a 45° measurement angle, a region with a depth of approximately 10 nm from the surface is detected. More specifically, the measurement is performed according to "(2) XPS Measurement" described later.

[0054] Porous adsorbent materials or protein adsorbent materials have pores for effectively removing impurities or proteins that are the targets of adsorption. The porous adsorbent materials or protein adsorbent materials of the present invention are not particularly limited in shape or size, as long as they have pores capable of adsorbing the target protein. If the average pore radius of the porous adsorbent material or protein adsorbent material is too small, the target protein will no longer enter the pores, making it unsuitable. Conversely, if the average pore radius is too large, the pore surface area per unit volume becomes small, and therefore, sometimes it is impossible to effectively adsorb the target protein. Therefore, the average pore radius value when measuring the porous adsorbent material or protein adsorbent material of the present invention using differential scanning calorimetry (DSC) is not particularly limited. As a lower limit, it is preferably 1 nm or more, more preferably 1.5 nm or more, and particularly preferably 2.0 nm or more. On the other hand, as an upper limit for the average pore radius when measuring the protein adsorbent material using DSC, it is preferably 100 nm or less, more preferably 40 nm or less, and particularly preferably 25 nm or less.

[0055] The average pore radius of porous adsorbents or protein adsorbents can be determined using differential scanning calorimetry (DSC). The porous adsorbent or protein adsorbent is quenched to -55°C, then heated to 5°C at a rate of 0.3°C / min and measured. The peak temperature of the resulting curve is taken as the melting point, and the average pore radius is calculated using the following formula.

[0056] Average pore radius [nm] = (33.30 - 0.3181 × (melting point of water - melting point calculated from the peak temperature of the curve) [°C]) / (melting point of water - melting point calculated from the peak temperature of the curve) [°C]

[0057] It should be noted that the above determination method is based on the record of Kazuhiro Ishikiriyama et al.; JOURNAL OF COLLOID AND INTERFACE SCIENCE, 171, 103-111, (1995).

[0058] In order to effectively adsorb and remove proteins as the target of adsorption using the porous adsorbent or protein adsorbent of the present invention, it is preferable that the porous adsorbent or protein adsorbent contains a large number of fine pores of optimal size. If the fine pores are too large or too small, the adsorption of proteins as the target of adsorption may sometimes be ineffective. For effective adsorption of proteins as the target of adsorption, when the protein adsorbent is observed and image analyzed using a scanning electron microscope (SEM), the ratio of the average pore diameter at the center to the average pore diameter near the surface is preferably 0.5 or more, more preferably 0.8 or more. On the other hand, when the protein adsorbent is observed and image analyzed using SEM, the ratio of the average pore diameter at the center to the average pore diameter near the surface is preferably 1.3 or less, more preferably 1.1 or less.

[0059] When comparing the average pore size near the surface of a porous adsorbent material or protein adsorbent material with the average pore size at its center, the cross-section of the porous adsorbent material or protein adsorbent material can be obtained using a scanning electron microscope (SEM) method. Using SEM at 50,000x magnification, the 640×480 pixel image was binarized using image processing software (ImageJ, developed by the National Institutes of Health), resulting in an image where the pores are black and the structural polymer portion is white.

[0060] Here, the cross-section of a porous adsorbent or protein adsorbent refers to the surface obtained by perpendicularly cutting the porous adsorbent or protein adsorbent with a surface that is in contact with a solution of biopharmaceutical raw materials, or a solution containing both the target protein and a non-target protein. For example, in the case of fibers, it refers to the surface cut perpendicular to the length direction. Image analysis is performed by observing the center and near the surface of the cross-section of the porous adsorbent or protein adsorbent. The pore radius near the surface refers to the pore radius of the surface of the porous adsorbent or protein adsorbent, i.e., the outer periphery of the cross-section. More specifically, when the ratio of the pore radius in a 225×225 pixel field of view 2 adjacent to the field of view 1 along a direction perpendicular to the surface to the pore radius in the 225×225 pixel field of view 1 in contact with the surface is less than 2, the 640×480 pixel field of view 1 that completely encompasses the field of view 1 is called the near surface. However, in the case of PMMA fibers, there are sometimes layers with very small pore radii on the surface, which are called dense layers. In order to adsorb and remove proteins that are the target of adsorption, it is appropriate, from the viewpoint that the pore radius can be optimized without deviation, to exclude part of this layer from the aforementioned "near the surface". Therefore, when the ratio of the pore radius of field of view 2 to the pore radius of field of view 1 is more than 2, the 640×480 pixel field of view that completely encompasses field of view 2 and completely excludes field of view 1 is referred to as the near surface.

[0061] Furthermore, the center of the longest straight line obtained by connecting two points on the surface is called the center. The average aperture is also calculated from the center.

[0062] In any of the above cases, the non-circular shape of the pore is assumed to be a perfect circle, and the pore radius is calculated from the pore area using the following formula.

[0063] The radius of the pores near the center or surface [nm] = (the area of ​​the pores near the center or surface [nm]) 2 ] / π) 1 / 2

[0064] Cut the porous adsorbent material or protein adsorbent material at any 5 points, and measure the pore radius at the center and near the surface of the resulting cross-sections. Calculate the average value of the pores and use it as the average pore diameter at the center or near the surface.

[0065] In this invention, the specific surface area of ​​the porous adsorbent material is defined as the area of ​​the surface in contact with the biopharmaceutical raw material solution containing the target protein, relative to the weight of the adsorbent material. For effective adsorption and removal of the target protein, a large specific surface area of ​​the porous adsorbent material is desirable; however, if it is too large, its strength decreases. For these reasons, a specific surface area of ​​0.001 m² is preferred.2 / g or more, more preferably 0.01m 2 / g or more, further preferably 0.05m 2 / g or more. On the other hand, 0.1m is preferred. 2 / g or less, more preferably 0.5m 2 / g or less.

[0066] One method for determining the amount of protein adsorbed by porous or protein-based adsorbents is to immerse the material in a solution containing both the target protein and other proteins, measure the amount of the target protein in the solution after a certain time, and subtract this amount from the amount of the target protein in the solution before immersion. Another method involves assembling the porous or protein-based adsorbent into a module or column, connecting a circuit, circulating the solution containing both the target and other proteins, measuring the amount of the target protein in the solution after a certain time, and subtracting this amount from the amount of the target protein in the solution before immersion.

[0067] There are no particularly limited methods for determining protein quality; examples include the Lowry method, dicaprylic acid method, ELISA, and immunoturbidimetry. Among these, ELISA and immunoturbidimetry are preferred due to their high specificity for the proteins to be adsorbed.

[0068] As described above, the layer disposed on at least one surface of the protein adsorbent material of the present invention comprises a polymer that inhibits the adhesion of biological components. Examples of polymers that inhibit the adhesion of biological components include, but are not limited to, polyvinylpyrrolidone, polyethylene glycol, and poly(2-methoxyethyl acrylate).

[0069] The polymer that inhibits the adhesion of biological components preferably contains nitrogen atoms. When the polymer that inhibits the adhesion of biological components contains nitrogen atoms, the form in which the nitrogen atoms exist is not particularly limited. From the viewpoint of effectively inhibiting the adhesion of biological components, the nitrogen atoms are preferably contained in the polymer in the form of hydrophilic groups such as amide groups.

[0070] The nonionic polymers or polymers that inhibit the adhesion of biological components used in this invention are preferably copolymers comprising hydrophilic and hydrophobic units. In this specification, "unit" refers to a repeating unit in a homopolymer or copolymer obtained by polymerizing monomers. Furthermore, a hydrophilic unit refers to a unit in which a polymer with a mass-average molecular weight of 10,000 to 1,000,000 is soluble in water when the polymer is manufactured solely using the monomer constituting that unit. "Soluble" means a solubility of more than 0.1 g relative to 100 g of water at 20°C.

[0071] In this specification, the hydrophobic unit is preferably a monocarboxylic acid vinyl ester unit.

[0072] Here, a monocarboxylic acid refers to a compound containing one carboxyl group and a hydrocarbon group bonded to the carbon atom of that carboxyl group, i.e., a compound represented by "R-COOH" (where R is a hydrocarbon group). The hydrocarbon group R in a monocarboxylic acid can be either an aliphatic hydrocarbon group or an aromatic hydrocarbon group. From the viewpoint of ease of synthesis, an aliphatic hydrocarbon group is preferred, and a saturated aliphatic hydrocarbon group is particularly preferred. Furthermore, from the viewpoint of the manufacturing cost of carboxylic acids, a saturated aliphatic hydrocarbon group is preferred to have a straight-chain structure or a branched structure, and a straight-chain structure is more preferred. Examples of monocarboxylic acids where the hydrocarbon group R is an aromatic hydrocarbon group include benzoic acid and its derivatives. Examples of monocarboxylic acids where the hydrocarbon group R is a saturated aliphatic hydrocarbon group include acetic acid, propionic acid, and butyric acid.

[0073] Saturated aliphatic hydrocarbon groups can be not only straight-chain structures such as ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl, but also branched structures such as isopropyl and tert-butyl, and cyclic structures such as cyclopropyl and cyclobutyl. Furthermore, ether bonds and ester bonds can be included within the aliphatic chain. It should be noted that the hydrogen atoms in the hydrocarbon group R can be substituted by any substituent. If the terminal hydrogen atom is replaced by anionic functional groups such as sulfonic acid groups, it may destabilize the structure of proteins that are not the target adsorbents, inducing their adhesion to the surface of the protein adsorbent material. Therefore, it is preferable that the terminal hydrogen atom is not replaced by anionic functional groups.

[0074] Having fewer carbon atoms in the hydrocarbon group R of a monocarboxylic acid is preferable in reducing the hydrophobicity of the monocarboxylic acid, minimizing hydrophobic interactions with proteins that are not the target for adsorption, and preventing adhesion. Therefore, when R is an aliphatic or aromatic hydrocarbon group, the number of carbon atoms is preferably 1 to 20, more preferably 1 to 9, and even more preferably 2 to 5. It should be noted that when R is a saturated aliphatic hydrocarbon group, the compound with 1 carbon atom is acetic acid, and the compound with 2 carbon atoms is propionic acid.

[0075] In this specification, "monocarboxylic acid vinyl ester unit" refers to a repeating unit in a homopolymer obtained by polymerizing monocarboxylic acid vinyl ester monomers. Preferably, such a monocarboxylic acid vinyl ester unit is a unit (repeating unit) represented by "-CH(OCO-R)-CH2-" (where R is a hydrocarbon group with 1 to 20 carbon atoms). Here, R is the same as described for the monocarboxylic acid, and preferred examples are also based on the above description.

[0076] The hydrocarbon group R with 2 to 20 carbon atoms in the monocarboxylic acid vinyl ester unit is preferably a saturated aliphatic hydrocarbon group or an aromatic hydrocarbon group. Among these, an aliphatic hydrocarbon group with 2 to 5 carbon atoms is preferred. Specific examples of monocarboxylic acid vinyl ester units where the hydrocarbon group R with 2 to 20 carbon atoms is a saturated aliphatic hydrocarbon group include vinyl propionate units, vinyl pentanoate units, vinyl decanoate units, and methoxyvinyl acetate units. From the perspective of avoiding excessive hydrophobicity, preferred examples include vinyl acetate units, vinyl propionate units, vinyl butyrate units, vinyl pentanoate units, vinyl pentanoate units, and vinyl hexanoate units. On the other hand, specific examples of monocarboxylic acid vinyl ester units where R is aromatic include vinyl benzoate units and their substitutes.

[0077] As a monomer constituting the hydrophobic unit, it contains at least a vinyl monocarboxylic acid ester, and may further contain units selected from acrylates, methacrylates, vinyl-ε-caprolactam, etc.

[0078] The monomer constituting the hydrophilic unit is more preferably a monomer with a solubility of more than 10 g relative to 100 g of water at 20°C. Examples of such monomers include vinyl alcohol monomers, acryloylmorpholine monomers, vinylpyridine monomers, vinylimidazolium monomers, and vinylpyrrolidone monomers. Among these, monomers having amide bonds, ether bonds, or ester bonds are preferred because their hydrophilicity is not excessive compared to monomers with carboxyl or sulfonic acid groups, and they are easier to balance with hydrophobic monomers. In particular, vinylacetamide monomers, vinylpyrrolidone monomers, and vinylcaprolactam monomers having amide bonds are more preferred, with vinylpyrrolidone monomers being further preferred due to the low toxicity of the polymer. Therefore, according to a preferred embodiment of the present invention, the polymer for inhibiting the adhesion of biological components contains vinylpyrrolidone units as hydrophilic units.

[0079] As a monomer constituting the hydrophobic unit, it contains at least a vinyl monocarboxylic acid ester, and may further contain units selected from acrylates, methacrylates, vinyl-ε-caprolactam, etc.

[0080] From the viewpoint of sufficiently inhibiting the adhesion of proteins that are not the target of adsorption, the number average molecular weight of the nonionic polymer or the polymer that inhibits the adhesion of biological components is preferably 1,000 or more, more preferably 5,000 or more. On the other hand, there is no particular limitation on the upper limit of the number average molecular weight of the polymer that inhibits the adhesion of biological components. From the viewpoint of avoiding a decrease in the porosity introduced into the protein adsorption material, it is preferably 1,000,000 or less, more preferably 500,000 or less, and even more preferably 100,000 or less. It should be noted that the number average molecular weight of the homopolymer or copolymer can be determined by gel permeation chromatography (GPC).

[0081] From the viewpoint of effectively suppressing the adhesion of proteins that are not the target of adsorption, the molar fraction of hydrophobic units in the copolymer containing the aforementioned hydrophilic and hydrophobic units is preferably 10% or more and 90% or less, more preferably 20% or more and 80% or less, and even more preferably 30% or more and 70% or less. In this case, the hydrophobic unit may be only a monocarboxylic acid vinyl ester unit, or it may further include other hydrophobic units. Setting the molar fraction of hydrophobic units to the above-mentioned upper limit is preferred in suppressing the overall increase in hydrophobicity of the copolymer, avoiding structural instability and modification of proteins that are not the target of adsorption, and thus preventing the adhesion of proteins that are not the target of adsorption. It should be noted that the above molar fraction is calculated, for example, by performing nuclear magnetic resonance (NMR) measurement and calculating based on the peak area ratio corresponding to each component. If the above molar fraction cannot be calculated by NMR measurement due to reasons such as peak overlap, the above molar fraction can be calculated by elemental analysis.

[0082] As described above, copolymers comprising vinyl ester monocarboxylate units and vinyl pyrrolidone units are particularly preferred as polymers for inhibiting the adhesion of biological components. In this case, the molar ratio of vinyl pyrrolidone units to vinyl ester monocarboxylate units is preferably 30:70 to 90:10, more preferably 40:60 to 80:20, and even more preferably 50:50 to 70:30.

[0083] Examples of unit arrangements in the aforementioned copolymers include block copolymers, alternating copolymers, and random copolymers. Among these, alternating copolymers or random copolymers are preferred from the viewpoint of minimizing the uneven distribution of hydrophilic and hydrophobic units in the copolymer as a whole. Random copolymers are more preferred from the viewpoint of ease of synthesis.

[0084] Methods for forming a layer comprising a polymer that inhibits the adhesion of biological components on at least one surface of a protein adsorbent material include: adding the polymer during the manufacture of the protein adsorbent material; and contacting the protein adsorbent material with the polymer after manufacture. From the viewpoint of having an appropriate thickness on at least one surface without making the layer containing the polymer that inhibits the adhesion of biological components excessively thick, methods that contact the protein adsorbent material with the polymer that inhibits the adhesion of biological components are preferred. Methods for contacting the protein adsorbent material with the polymer that inhibits the adhesion of biological components include: immersing the protein adsorbent material in a solution of the polymer that inhibits the adhesion of biological components; introducing the polymer solution into the protein adsorbent material; and blowing the polymer solution onto the protein adsorbent material by spraying or the like. From the viewpoint of uniformly applying the polymer that inhibits the adhesion of biological components to the surface of the protein adsorbent material, methods that immerse the protein adsorbent material in a solution of the polymer that inhibits the adhesion of biological components or introducing the polymer solution into the protein adsorbent material are preferred.

[0085] When a protein adsorbent is impregnated with a polymer solution that inhibits the adhesion of biological components, or when a polymer solution that inhibits the adhesion of biological components is introduced into the protein adsorbent, if the concentration of the polymer in the solution is too low, a sufficient amount of the polymer will not be introduced to the surface. Therefore, the concentration of the polymer in the solution that inhibits the adhesion of biological components is preferably 10 ppm or more, more preferably 100 ppm or more. If the concentration is too high, there is a concern that the amount of leached material from the protein adsorbent will increase, the polymer will penetrate into the pores, the thickness of the layer containing the polymer that inhibits the adhesion of biological components will increase, and the adsorption performance will decrease. Therefore, the concentration of the polymer in the solution that inhibits the adhesion of biological components is preferably 100,000 ppm or less, more preferably 10,000 ppm or less, and even more preferably 1,000 ppm or less.

[0086] Water is preferably used as the solvent for preparing the polymer solution that inhibits the adhesion of biological components. However, if the polymer used to inhibit the adhesion of biological components has low solubility relative to water, it can be dissolved in an organic solvent that does not dissolve the protein adsorbent, or in a mixture of an organic solvent miscible with water and water that does not dissolve the protein adsorbent. Examples of organic solvents that can be used include, but are not limited to, alcohol-based solvents such as methanol, ethanol, or propanol.

[0087] As a method for forming a layer of a polymer containing an inhibitor of bio-adhesion in a protein adsorbent material, it is ideal to immobilize the polymer onto the surface of the protein adsorbent material using chemical methods. There are no particular limitations on the method for immobilizing the polymer onto the surface of the protein adsorbent material using chemical methods; examples include: irradiating the protein adsorbent material with radiation after contacting it with the polymer; and introducing reactive groups such as amino or carboxyl groups into both the protein adsorbent material and the polymer, causing a reaction between them. Examples of methods for introducing reactive groups onto the surface of the protein adsorbent material include: polymerizing monomers with reactive groups to obtain a material with reactive groups on its surface; and introducing reactive groups after polymerization through ozone treatment or plasma treatment.

[0088] Furthermore, when using irradiation, the radiation source can be alpha rays, beta rays, gamma rays, X-rays, ultraviolet rays, and electron beams. Irradiation is carried out under the following conditions: contacting the protein adsorbent material with a solution containing a polymer that inhibits the adhesion of biological components; or, after introducing the polymer that inhibits the adhesion of biological components onto the surface of the protein adsorbent material, replacing it with a solution that does not contain the polymer; or drying the protein adsorbent material. Using this method, both the immobilization of the polymer that inhibits the adhesion of biological components and the sterilization of the protein adsorbent material can be achieved, which is therefore preferable. In this case, the radiation dose is preferably 15 kGy or more, more preferably 25 kGy or more. On the other hand, if the radiation dose is too high, it promotes the deterioration and decomposition of the polymer; therefore, the radiation dose is preferably 100 kGy or less.

[0089] To suppress cross-linking reactions during radiation exposure, antioxidants can be used. Antioxidants are substances that readily donate electrons to other molecules; examples include water-soluble vitamins such as vitamin C; polyphenols; and alcoholic solvents such as methanol, ethanol, or propanol, but are not limited to these. These antioxidants can be used alone or in combination of two or more. When using antioxidants in protein adsorbent materials, safety must be considered; therefore, antioxidants with low toxicity, such as ethanol and propanol, are suitable.

[0090] In addition, the same method described above can be used to apply nonionic polymers to the surface of porous materials.

[0091] The porous adsorbent or protein adsorbent of the present invention exhibits excellent protein adsorption performance, and therefore can be applied to the pretreatment process of antibody recovery or antibody recovery using a protein A column. The size of the antibody is typically considered to be 10–15 nm. When the pore size of the protein adsorbent of the present invention is smaller than the antibody size, the antibody is not adsorbed, and the adsorption and removal of HCP, antibody decomposition products, components derived from the culture medium, DNA, viruses, free protein A, etc., smaller than the antibody, can be achieved based on the size difference.

[0092] The porous adsorbent material of the present invention has high separation performance, and therefore can be used in the process of purifying biopharmaceuticals from biopharmaceutical raw material solutions to recover the active ingredients of biopharmaceuticals. When the biopharmaceutical is an antibody, antibody loss will occur in subsequent processes; therefore, the antibody recovery rate after treating the biopharmaceutical raw material solution with the porous adsorbent material is preferably as high as possible. Specifically, the antibody recovery rate is preferably 85% or more, and more preferably 90% or more.

[0093] On the other hand, it is desirable to remove as much of the impurities represented by HCP as possible. Therefore, the residual rate of HCP after treating the biopharmaceutical raw material solution with porous adsorption materials is preferably as low as possible. Specifically, it is preferably 60% or less, more preferably 50% or less, and even more preferably 40% or less. The separation ratio, obtained by dividing the antibody recovery rate by the residual rate of HCP, becomes an indicator of the separation selectivity of the porous adsorption material. The separation ratio is preferably 1.5 or more, more preferably 1.8 or more, and even more preferably 2 or more. The method shown in the examples can be used as the method for calculating the antibody recovery rate, the residual rate of HCP, and the separation ratio.

[0094] In the manufacturing process of antibody proteins, namely monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, and immunoglobulins, which are used as a type of biopharmaceutical, when the protein adsorbent material of the present invention is used in the pretreatment process of the antibody recovery and separation process from the biopharmaceutical raw material solution or the antibody recovery process using a protein A column, excessive adsorption of antibodies onto the protein adsorbent material leads to antibody loss. Therefore, the antibody adsorption amount is preferably 100 mg / m³. 2 Below. On the other hand, since the aim is to remove as much of the inclusions represented by HCP as possible, the adsorption capacity of HCP is preferably 100 mg / m³. 2 The above applies. Since a low adsorption capacity of the antibody and a high adsorption capacity of HCP are required, the protein adsorption ratio, obtained by dividing the HCP adsorption capacity by the antibody adsorption capacity, can be considered an indicator of high purification efficiency for protein adsorbent materials. The preferred protein adsorption ratio is 2 or higher.

[0095] The antibody adsorption capacity of the porous adsorbent or protein adsorbent of the present invention is 100 mg / m³. 2 When the separation ratio is 1.5 or higher, it is very useful as a material for adsorbing and removing HCP, which is an impurity, from the culture supernatant of antibody-producing cells. A separation ratio of 1.0 or higher is more preferred, and 2.0 or higher is even more preferred. If the separation ratio is within the above range, the amount of HCP per unit antibody quantity, which is an indicator of HCP removal efficiency, becomes a negligible level of tens of ppm when transferred to downstream processes such as ion chromatography. Furthermore, if the antibody adsorption amount is within the above range, clogging of the adsorption material's mesh can be prevented.

[0096] For example, solid PMMA fibers with an average pore radius of 5 nm were impregnated in an aqueous solution containing a vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units and a number-average molecular weight of 68,000) dissolved in ethanol at a concentration of 100 ppm and 500 ppm, and then irradiated with 25 kGy of gamma rays. The resulting material was then measured with a surface area of ​​0.03 m². 2 Samples were taken and placed into capped tubes. 6.75 mL of culture supernatant from Chinese hamster ovary (CHO) cells (antibody concentration 3.63 mg / mL, HCP concentration 1.46 mg / mL) adjusted to pH 7.4 was added, and the tubes were capped. The tubes were then incubated at room temperature with shaking for 6 hours. The culture supernatant was then sampled, and the antibody concentration was determined using immunoturbidimetry. The HCP concentration in the culture supernatant was determined using a CHO Host Cell Proteins 3rd Generation (Cygnus Technologies) ELISA method. The adsorption amounts of antibody, HCP, and protein were calculated using the following formulas.

[0097] Antibody adsorption capacity (mg / m 2 = (Antibody concentration before adsorption × liquid volume) - (Antibody concentration after adsorption × liquid volume)) ÷ 0.03

[0098] HCP adsorption capacity (mg / m³) 2 = (HCP concentration before adsorption × liquid volume) - (HCP concentration after adsorption × liquid volume)) ÷ 0.03

[0099] Protein adsorption ratio = HCP adsorption amount ÷ antibody adsorption amount.

[0100] In this case, the adsorption capacity of antibodies larger than the pore size is 66 mg / m². 2 The adsorption capacity of HCP with a smaller pore size is 148 mg / m³. 2The protein adsorption ratio is 2.24. Since this meets the aforementioned preferred criteria, the adsorption capacity for proteins that are not the target adsorption material but have a pore size larger than that of the protein adsorbent material of the present invention is 100 mg / m³. 2 The following is true, and the adsorption capacity of HCP is 100 mg / m³. 2 When the protein adsorption ratio is 2 or higher, it is preferred, especially for antibody recovery processes in the manufacturing process of antibody drugs.

[0101] In addition, the antibody recovery rate was 92%, the HCP residue rate was 55%, and the separation ratio was 1.7, which can be interpreted as an excellent adsorbent material for proteins, which are impurities used to recover the active ingredients of biopharmaceuticals from biopharmaceutical raw material solutions.

[0102] The separation ratio of the above materials is 1.7, and therefore, the antibody adsorption capacity is 66 mg / m³. 2 Therefore, it can be said that it is useful as a material for adsorbing and removing HCP as an impurity from the culture supernatant of antibody-producing cells.

[0103] The typical manufacturing process for antibody drugs includes culturing antibody-producing cells, separating the cells from the antibodies, recovering and purifying the antibodies, inactivating the virus, and removing the virus. In the cell-antibody separation process, centrifugation and deep filtration are used. Antibody recovery primarily utilizes protein A columns, which are obtained by immobilizing protein A, which specifically adsorbs antibodies. Additionally, in the purification process, anion exchange columns and cation exchange columns are used to remove the animal cell-derived proteins (HCPs) used to produce the antibodies. In the virus inactivation process, treatment at low pH levels (below 4) is common.

[0104] In the case of antibody-based biopharmaceuticals, the biopharmaceutical raw material solution also contains antibody aggregates and decomposition products as impurities. The molecular weight of antibody decomposition products varies depending on the site of decomposition, mainly falling within the range of 10 kDa to 110 kDa, which is close to the molecular weight distribution of HCP. Therefore, they can be adsorbed and removed using the porous adsorption material of this invention. Thus, the porous adsorption material of this invention can adsorb antibody decomposition products other than the antibody itself, which are difficult to remove using a protein A column, thereby providing antibody-based pharmaceuticals with fewer impurities and greater safety.

[0105] In manufacturing processes using protein A columns, if protein A becomes free from the carrier immobilized with protein A and dissolves along with the antibody, the free protein A may potentially have adverse effects on organisms. The porous adsorption material of this invention does not utilize antibody-binding ligands like protein A; therefore, it is impossible to include protein A in the final antibody drug product, thus providing a safe antibody drug with fewer side effects.

[0106] The separation column for refining biopharmaceuticals of the present invention contains the porous adsorption material of the present invention.

[0107] The shape of the shell of the separation column for biopharmaceutical purification according to the present invention can be exemplified by open-ended shapes such as square cylinders, hexagonal cylinders, and cylindrical cylinders, with a cylindrical shape being preferred, and a cylinder with a perfectly circular cross-section being particularly preferred. This is because by eliminating the corners of the shell, blood stagnation at the corners can be suppressed. Furthermore, by making both sides open, pressure loss can be minimized, preventing the flow path of the processed liquid from becoming turbulent. Additionally, the shell is preferably made of plastic, metal, or the like. From the viewpoints of cost, moldability, and weight, plastic is suitable for use. In the case of plastic, thermoplastic resins with excellent mechanical strength and thermal stability are used, for example. Specific examples of such thermoplastic resins include polycarbonate resins, polyvinyl alcohol resins, cellulose resins, polyester resins, polyarylate resins, polyimide resins, cyclic polyolefin resins, polysulfone resins, polyethersulfone resins, polyolefin resins, polystyrene resins, and mixtures thereof. Among these, polypropylene, polystyrene, polycarbonate, and their derivatives are preferred in terms of the desired formability and radiation resistance of the shell. This is because resins with excellent radiation resistance are preferred when subjected to radioactive irradiation during sterilization. The resin is manufactured by injection molding based on a mold and by machining the raw material.

[0108] When the porous adsorbent of the present invention is incorporated into the separation column for the purification of biopharmaceuticals of the present invention, the porous adsorbent is preferably arranged in a straight line. This is because the liquid to be treated flows along the porous adsorbent, thus reducing the likelihood of turbulence and facilitating the even distribution of the liquid within the column. Furthermore, it suppresses flow resistance, and is particularly advantageous when treating high-viscosity solutions containing proteins such as antibodies, as it enables effective protein adsorption.

[0109] The method for manufacturing the biopharmaceutical of the present invention includes: a step of preparing a biopharmaceutical raw material solution, and a step of contacting the biopharmaceutical raw material solution with a porous adsorption material.

[0110] The method for manufacturing the biopharmaceutical of the present invention may include other purification methods besides the steps of preparing the biopharmaceutical raw material solution and contacting the biopharmaceutical raw material solution with a porous adsorbent material. Other purification methods are not particularly limited as long as they are suitable for manufacturing pharmaceuticals, and examples include chromatography (e.g., anion exchange chromatography, cation exchange chromatography, and mixed chromatography), virus inactivation, and virus removal.

[0111] In the method for manufacturing biopharmaceuticals of the present invention, a porous adsorbent material for adsorbing and removing impurities from a biopharmaceutical raw material solution and a porous hollow fiber membrane for separating and removing impurities are prepared, wherein the biopharmaceutical raw material solution is continuously processed using the aforementioned porous adsorbent material and the aforementioned porous hollow fiber membrane.

[0112] In the method for manufacturing the biopharmaceutical of the present invention, there is no particular limitation on the manner in which the aforementioned porous adsorbent material or the aforementioned porous hollow fiber membrane is housed. From the viewpoint of ease of connection and ease of taking the purified solution, it is preferable to use a column or module in which the aforementioned porous adsorbent material or the aforementioned porous hollow fiber membrane is housed.

[0113] "Continuously" means that the outlet of the purified solution discharged from the column or module containing the aforementioned porous adsorbent material or porous hollow fiber membrane is connected to the next module or column containing the aforementioned porous hollow fiber membrane or porous adsorbent material. It should be noted that this can be in the form of merging the processed liquid from other modules or columns containing the porous hollow fiber membrane or porous adsorbent material into a space between modules or columns containing the porous hollow fiber membrane and those containing the porous adsorbent material, or it can be in the form of branching the processed liquid from between modules or columns containing the porous hollow fiber membrane and those containing the porous adsorbent material into other modules or columns containing the porous hollow fiber membrane or porous adsorbent material. The processing order of the aforementioned porous adsorbent material or the aforementioned porous hollow fiber membrane is not particularly limited, but if the aforementioned porous adsorbent material is used for adsorption first, the separation performance of the aforementioned porous hollow fiber membrane may be affected due to changes in the composition of the biopharmaceutical raw material solution. Therefore, it is preferable to first perform separation based on the porous hollow fiber membrane.

[0114] It should be noted that the aforementioned module with built-in porous hollow fiber membrane can be multi-stage. That is, multiple such modules are arranged, with the filtrate outlet of the previous stage module connected to the next stage module. The treated liquid is sequentially sent to the multi-stage modules for repeated separation. Through multi-stage arrangement, the recovery rate of substances with pore sizes larger than those of the porous hollow fiber membrane is improved.

[0115] Examples of ways to allow biopharmaceutical feedstock solutions to flow into porous hollow fiber membranes include: total filtration, in which the entire biopharmaceutical feedstock solution passes through the membrane and the biopharmaceutical residue is recovered by washing; and cross-flow, in which the biopharmaceutical feedstock solution is circulated multiple times while the liquid is added during filtration to purify the biopharmaceutical. From the perspective of improving the recovery rate by preventing biopharmaceutical residues from remaining in the membrane, the cross-flow method is preferred.

[0116] The following are examples of the fabrication of protein adsorbent materials, which serve as representative examples. The fabrication of the porous adsorbent material described in this invention will be explained below.

[0117] [The production of solid fibers]

[0118] A spinning solution is prepared by dissolving a polymer in a solvent. The lower the solution concentration (the concentration of substances other than the solvent in the solution), the greater the fiber pore size can be increased. Therefore, by appropriately setting the solution concentration, the pore size / pore volume can be controlled. Furthermore, the pore size / pore volume can also be controlled by using a polymer with negatively charged groups. From this perspective, a solution concentration of 30% by mass or less is preferred in this invention, more preferably 27% by mass or less, and even more preferably 24% by mass or less. Additionally, when using a polymer, for example, having methacrylic acid-p-styrene sulfonic acid as a negatively charged group, the proportion of the polymer with methacrylic acid-p-styrene sulfonic acid present in the total polymer is preferably 10 mol% or less.

[0119] If the viscosity of the spinning solution is too low, the solution will have high fluidity and be difficult to maintain the target shape. Therefore, the lower limit of the solution viscosity is 10 poise or more, more preferably 90 poise or more, further preferably 400 poise or more, and particularly preferably 800 poise or more. On the other hand, if the viscosity is too high, the pressure loss during solution ejection will increase, resulting in reduced ejection stability or difficulty in mixing the solution. Therefore, the upper limit of the solution viscosity at the temperature at the spinning tube head is 100,000 poise or less, more preferably 50,000 poise or less.

[0120] Fibers are obtained by spraying the raw liquid from the nozzle and passing it through a dry air section of a certain distance, and then spraying it into a coagulation bath containing undesirable solvents such as water or non-solvents.

[0121] The shape of the fiber cross-section is not particularly limited. When using fibers with irregular cross-sectional shapes as protein adsorbent materials, it is preferable to set them to be mixed with round or elliptical fibers.

[0122] Both post-blending and spinning blending methods, which are currently known, can be used as methods for manufacturing blended fibers. Examples of post-blending methods include: feeding fibers into the washing and winding processes during spinning and blending; blending through air interlacing; blending through twisting, doubling, and straightening; blending through interlacing; blending through cross-knitting; and dispersing the fibers in a liquid and recovering them, but these are not limited to these methods. Additionally, examples of spinning blending methods include composite spinning. Composite spinning includes methods such as: simultaneously ejecting and winding multiple yarns from the same nozzle with multiple nozzles; and using a single-nozzle nozzle to mix nozzles with different nozzle shapes in a nozzle ejection block, thereby simultaneously ejecting and winding yarns with different cross-sectional shapes, etc. Furthermore, post-blending is more prone to localized unevenness compared to spinning blending methods. When observing the fiber cross-sectional state, localized unevenness sometimes occurs, posing a problem for fiber quality stability. In the case of fiber blending during spinning, unevenness is less likely to occur, and the quality is more stable, which is therefore preferred. Furthermore, by appropriately changing the structure and configuration of the nozzle, the combination of fiber shapes and the blending ratio can be easily changed, which is also advantageous. It should be noted that additives, etc., can be added to the extent that they do not impair the function / effect of this invention.

[0123] The dry section refers to the portion of the spinning solution that travels in the air from the nozzle until it comes into contact with a poor solvent or until its structure is completely fixed by cooling. During the structural fixation of the spinning solution, the area near the surface of the solution is in a high-energy state. Therefore, it can be assumed that upon contact with a poor solvent or moisture contained in the air, the supporting components, such as polymers, aggregate to form a fiber surface. Therefore, it is necessary to establish a porous structure to some extent in the dry section until the spinning solution contacts the poor solvent. Specifically, it is important to: rapidly induce phase separation after the solution is ejected, and sufficiently allow the pore structure to grow / expand before contact with the poor solvent; increase the viscosity of the solution by cooling the fiber in the dry section, and suppress aggregation by reducing the migration of the supporting components. To achieve this, it is important to ensure a sufficient residence time in the dry section. Therefore, the residence time is 0.05 seconds or more, preferably 0.20 seconds or more, and more preferably 0.40 seconds or more. The residence time is calculated according to the following formula.

[0124] Residence time (seconds) = Dry length (m) / Winding speed (m / second)

[0125] Furthermore, when the temperature of the ejected fibers decreases in the dry section, causing rapid structural fixation such as gelation and solidification, cold air can be blown into the dry section to promote gelation. While the exact mechanism is not yet fully understood, increasing the cold air velocity and cooling efficiency can expand the open area of ​​the fiber surface and the pore size near the fiber's outer periphery. The spinning solution ejected from the nozzle solidifies in a coagulation bath. The coagulation bath typically contains a coagulant such as water or alcohol, or a mixture of these with solvents constituting the spinning solution. Water is most commonly used. Furthermore, the pore size can be altered by controlling the temperature of the coagulation bath. Since the pore size can be affected by factors such as the type of spinning solution, the temperature of the coagulation bath is appropriately selected. Generally, increasing the coagulation bath temperature can increase the pore size. The exact mechanism is not yet fully understood, but it is thought that the competitive reaction between desolvation from the solution and solidification shrinkage leads to rapid desolvation in the high-temperature bath, resulting in solidification and fixation before the fiber shrinks internally. However, if the coagulation bath temperature becomes too high, the pore size becomes too large, thus affecting the specific surface area, strength, and elongation, and increasing non-specific adsorption. Therefore, for example, when the fiber contains PMMA, the coagulation bath temperature is preferably 90°C or lower, more preferably 75°C or lower, and particularly preferably 65°C or lower. On the other hand, if the coagulation temperature is too low, the pore size shrinks, making it difficult for adsorbed substances to diffuse into the fine pores. Therefore, as a lower limit, it is preferably 12°C or higher, more preferably 20°C or higher.

[0126] Next, the fibers are washed to remove the solvent adhering to the coagulated fibers. The method of washing the fibers is not particularly limited, but it is preferable to use a method that involves passing the fibers through a multi-stage water bath (called a water bath). The temperature of the water in the water bath is determined according to the properties of the polymer constituting the fibers. For example, in the case of fibers containing PMMA, 30–50°C is used.

[0127] In addition, to maintain the pore size after washing, a process can be added to impart moisturizing ingredients to the fibers. These moisturizing ingredients refer to components that can retain fiber moisture or prevent the fibers from losing moisture in the air. Examples of representative moisturizing ingredients include glycerin and its aqueous solutions.

[0128] To improve the dimensional stability of highly shrinkable fibers after washing and application of moisturizing agents, they can be subjected to a process called a heat treatment bath filled with a heated aqueous solution of moisturizing agents. In this heat treatment bath, the fibers are subjected to heat and shrink, making them less prone to shrinkage in subsequent processes and stabilizing the fiber structure. The heat treatment temperature varies depending on the fiber raw material, but for fibers containing PMMA, 50°C or higher is preferred, and 80°C or higher is more preferred. Furthermore, a temperature of 95°C or lower is preferred, and 87°C or lower is more preferred.

[0129] There are no particular limitations on the method for fabricating a separation column for biopharmaceutical purification using the obtained porous adsorbent material; one example is shown below. First, the porous adsorbent material is cut to the necessary length and loaded into the shell in a straight line along the axial direction of the shell. Then, the two ends of the porous adsorbent material are cut using a cutter or similar means, allowing the porous adsorbent material to fall into the shell. Mesh filters, cut to the same diameter as the inner diameter, are installed at the inlet and outlet of the treated liquid on both ends of the column. Finally, inlets and outlets for the treated liquid, referred to as top covers, are installed at both ends of the shell, thus obtaining the separation column for biopharmaceutical purification.

[0130] Example

[0131] The following examples and comparative examples are described in detail, but the present invention is not limited to them.

[0132] (1) TOF-SIMS measurement (determination of the thickness of a layer containing a non-ionic polymer or a polymer that inhibits the adhesion of biological components)

[0133] The sample (one fiber) was embedded in resin, and a cross-section was prepared using a slicer. The measurements were performed using a TOF-SIMS5 (manufactured by ION-TOF) under the following conditions.

[0134] Primary ion: Bi3++

[0135] Primary ion acceleration voltage: 25kV

[0136] Pulse width: 125.0ns

[0137] Secondary ion polarity: negative

[0138] Number of scans: 96

[0139] Cycle time: 200μs

[0140] Grating size: 50×50μm 2

[0141] Mass range (m / z): 0~1500

[0142] <Determination of Layer Thickness 1>

[0143] Based on the obtained mass m / z spectrum, spectral profiles were constructed for any three locations on the solid fiber cross-section, and the thickness of the layer containing the polymer that inhibits the adhesion of biological components was calculated to two significant figures. The average value of the three locations was taken as the thickness of the layer containing the polymer that inhibits the adhesion of biological components.

[0144] <Determination of Layer Thickness 2>

[0145] Based on the obtained mass m / z spectrum, spectral profiles were constructed for any one location on the solid fiber cross-section. The CN values ​​representing polymers that inhibit the adhesion of biological components were then analyzed. - +CNO - A line segment is drawn from the flat part of the outline. The distance between the two points where the line segment intersects the two ends of the peak of the polymer that inhibits the adhesion of biological components is taken as the thickness of the layer containing the polymer that inhibits the adhesion of biological components, and is expressed in two significant figures.

[0146] It should be noted that the vinylpyrrolidone / vinyl propionate random copolymers (40% molar fraction of vinyl propionate units and 68,000 number-average molecular weight), vinylpyrrolidone / vinyl hexanoate random copolymers (40% molar fraction of vinyl hexanoate units and 68,000 number-average molecular weight), and vinylpyrrolidone / vinyl propionate random copolymers (40% molar fraction of vinyl propionate units and 100,000 number-average molecular weight) used in the following embodiments are equivalent to the polymers mentioned in this invention that inhibit the adhesion of biological components. In addition, they are nonionic polymers.

[0147] (2) XPS determination (the proportion of nitrogen atoms relative to all atoms 100 (atomic percentage) (atomic percentage))

[0148] After rinsing the sample with ultrapure water, it was dried at room temperature and 0.5 Torr for 10 hours and then used for determination.

[0149] [Measurement Conditions]

[0150] Device: Quantera SXM (manufactured by PHI)

[0151] Excitation X-rays: monochromatic AlKα1,2 rays (1486.6 eV)

[0152] X-ray diameter: 100 μm

[0153] Photoelectronic detection angle: 45° (slope of the detector relative to the sample surface)

[0154] As the atomic mass of nitrogen, the proportion of the peak area appearing near 400 eV of N1s to all elements (since hydrogen atoms cannot be detected, this includes all elements except hydrogen atoms) is calculated to determine the atomic mass (atomic percentage).

[0155] (3) Determination of average pore radius

[0156] The freezing point depression was determined by measuring the capillary aggregation of water within the pores using differential scanning calorimetry (DSC). Specifically, the sample was quenched to -55°C, then heated to 5°C at a rate of 0.3°C / min and measured. The peak temperature of the resulting curve was taken as the melting point, and the average pore radius was calculated using the following formula.

[0157] Average pore radius [nm] = (33.30 - 0.3181 × melting point decrease [°C]) / melting point

[0158] The measuring apparatus used was the DSC Q1001 manufactured by TA Instruments.

[0159] It should be noted that the melting point decrease is the difference obtained by subtracting the peak temperature of the aforementioned curve from the melting point of water (0°C).

[0160] (4) Determination of average pore size

[0161] The surface of the sample was observed using a scanning electron microscope (SEM) (S-5500, manufactured by Hitachi High-Technologies), and image analysis was performed. The SEM was used to observe the sample at 50,000x magnification. The 640×480 pixel images were binarized using image processing software (ImageJ, developed by the National Institutes of Health), resulting in images where the pores were black and the structural polymer areas were white.

[0162] Observe the center and near the surface of the sample cross-section and perform image analysis. If the ratio of the aperture radius in a 225×225 pixel field of view 2 (perpendicular to the surface and adjacent to field of view 1) to the aperture radius in a 225×225 pixel field of view 1 (contacting the surface) is less than 2, then a 640×480 pixel field of view completely encompassing field of view 1 is considered near the surface. If the ratio of the aperture radius in field of view 2 to the aperture radius in field of view 1 is more than 2, then a 640×480 pixel field of view completely encompassing field of view 2 and completely excluding field of view 1 is considered near the surface. The center of the longest straight line connecting the two points on the surface is taken as the center. Assuming the aperture is a perfect circle, the aperture radius is calculated based on the aperture area using the following formula.

[0163] The radius of the pores near the center or surface [nm] = (the area of ​​the pores near the center or surface [nm]) 2 ] / π) 1 / 2

[0164] The pore radius is determined by measuring the cross-sections obtained from cutting the sample at five arbitrary points, both at the center and near the surface. The average value of these values ​​is then used as the average pore diameter near the center or surface. The ratio of the average pore diameter at the center to the average pore diameter near the surface is calculated using the following formula.

[0165] The ratio of the average aperture at the center to the average aperture near the surface = average aperture at the center / average aperture near the surface.

[0166] (5) Adsorption performance evaluation

[0167] Using a single-edged blade with a film area of ​​0.03m² 2 Samples were collected by drying them on a paper towel and then placing them into a capped plastic tube. 6.75 mL of CHO cell culture supernatant (hereinafter referred to as "culture supernatant"), adjusted to pH 7.4 by adding 0.6 N hydrochloric acid, was added, and the tube was capped. The tube was then agitated at room temperature using a seed shaker (TAITEC). Six hours after adding the culture supernatant, samples were taken to determine the antibody and HCP concentrations.

[0168] Antibody concentration was determined using immunoturbidimetry. HCP concentration was determined using CHO Host Cell Proteins 3rd Generation (Cygnus Technologies) and ELISA. The following formulas were used to calculate antibody adsorption, HCP adsorption, antibody recovery, HCP residue, protein adsorption ratio, and separation ratio.

[0169] Antibody adsorption capacity (mg / m 2 = (Antibody concentration before adsorption × liquid volume) - (Antibody concentration after adsorption × liquid volume)) ÷ 0.03

[0170] HCP adsorption capacity (mg / m³) 2 = (HCP concentration before adsorption × liquid volume) - (HCP concentration after adsorption × liquid volume)) ÷ 0.03

[0171] Protein adsorption ratio = HCP adsorption amount ÷ Antibody adsorption amount

[0172] Antibody recovery rate (%) = (Antibody concentration after adsorption × liquid volume) ÷ (Antibody concentration before adsorption × liquid volume)) × 100

[0173] HCP residual rate (%) = (HCP concentration after adsorption × liquid volume) ÷ (HCP concentration before adsorption × liquid volume)) × 100

[0174] Separation ratio = Antibody recovery rate ÷ HCP residual rate

[0175] It should be noted that the antibody concentration in the culture supernatant used before adsorption was 3.63 mg / mL and the HCP concentration was 1.46 mg / mL.

[0176] As indicators of adsorption performance, the antibody and HCP adsorption amounts, protein adsorption ratios, and separation ratios are used. When purifying antibodies from culture supernatants using the porous adsorption material or protein adsorption material of this invention, a low adsorption amount of the target antibody is required, while a high adsorption amount of impurities represented by HCP is desired. Therefore, it can be said that the antibody adsorption amount, HCP adsorption amount, the protein adsorption ratio obtained by dividing the HCP adsorption amount by the antibody adsorption amount, and the separation ratio obtained by dividing the antibody recovery rate by the HCP residue rate are indicators of high purification efficiency for protein adsorption materials.

[0177] (Example 1)

[0178] [Manufacturing of protein adsorbent materials with fine pores]

[0179] 31.7 parts by weight of syn-PMMA with a mass average molecular weight of 400,000, 31.7 parts by weight of syn-PMMA with a mass average molecular weight of 1,400,000, 16.7 parts by weight of iso-PMMA with a mass average molecular weight of 500,000, 20 parts by weight of PMMA copolymer containing 1.5 mol% sodium p-styrene sulfonate and with a molecular weight of 300,000, and 376 parts by weight of dimethyl sulfoxide were mixed and stirred at 110°C for 8 hours to prepare a spinning solution. The resulting spinning solution was sprayed into the air at a rate of 1.1 g / min through cross-shaped and elliptical tubes (tube ratio = 17:7) kept at 92°C. After traveling 380 mm in the air, it was introduced into a coagulation bath, where it passed through to obtain cross-shaped and elliptical solid fibers. Water was used in the coagulation bath at a temperature of 42°C. After washing the fibers, they were introduced into a bath containing an aqueous solution of 70% by mass glycerin as a humectant. The fibers were then subjected to a heat treatment bath at 84°C to remove excess glycerin, and then wound at a speed of 16 m / min. This was used as manufacturing example 1.

[0180] Manufacturing Example 1 was measured using an electronic balance and rinsed 10 times while changing the water. After removing the moisture on a paper towel, it was placed into a capped plastic tube. An aqueous solution containing 50 ppm of vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units, number average molecular weight 68,000) and 500 ppm of ethanol was added to the plastic tube, and the tube was irradiated with 25 kGy of gamma rays. After gamma ray irradiation, the copolymer aqueous solution was removed, and the tube was rinsed 10 times while changing the water. This was used as Example 1.

[0181] Regarding Example 1, the thickness of the layer containing the polymer that inhibits the adhesion of biological components, the ratio of nitrogen atoms to 100% of total atoms, the average pore radius, the average pore size, and the adsorption performance were determined using the aforementioned method. The results are shown in Table 1.

[0182] (Example 2)

[0183] Except that the concentration of the vinylpyrrolidone / vinyl propionate random copolymer of Example 1 (40% molar fraction of vinyl propionate units and number-average molecular weight of 68,000) was set to 100 ppm, the same treatment as in Example 1 was performed. This was used as Example 2. The results are shown in Table 1.

[0184] (Example 3)

[0185] Except that the concentration of the vinylpyrrolidone / vinyl propionate random copolymer of Example 1 (40% molar fraction of vinyl propionate units and number-average molecular weight of 68,000) was set to 200 ppm, the same treatment as in Example 1 was performed. This was designated as Example 3. The results are shown in Table 1.

[0186] (Example 4)

[0187] Instead of the aqueous solution prepared in Example 1 with a vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units, number average molecular weight of 68,000) at a concentration of 100 ppm and an ethanol concentration of 500 ppm, an aqueous solution prepared in Example 4 with a vinylpyrrolidone / vinyl hexanoate random copolymer (40% molar fraction of vinyl hexanoate units, number average molecular weight of 68,000) at a concentration of 200 ppm and an ethanol concentration of 500 ppm was used. All other things being equal to Example 1, this was used as Example 4. The results are shown in Table 1.

[0188] (Example 5)

[0189] Instead of the aqueous solution prepared in Example 1 with a vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units, number average molecular weight 68,000) at a concentration of 100 ppm and an ethanol concentration of 500 ppm, an aqueous solution prepared in the same manner as in Example 1 was used, except for this difference. This is referred to as Example 5. The results are shown in Table 1.

[0190] (Example 6)

[0191] Except that the concentration of the vinylpyrrolidone / vinyl propionate random copolymer of Example 1 (40% molar fraction of vinyl propionate units and number-average molecular weight of 68,000) was set to 10 ppm, the same treatment as in Example 1 was performed. This was designated as Example 6. The results are shown in Table 1.

[0192] (Example 7)

[0193] Except that the concentration of the vinylpyrrolidone / vinyl propionate random copolymer of Example 1 (40% molar fraction of vinyl propionate units and number-average molecular weight of 68,000) was set to 1,000 ppm, the same treatment as in Example 1 was performed. This was designated as Example 7. The results are shown in Table 1.

[0194] (Comparative Example 1)

[0195] Instead of the aqueous solution prepared in Example 1 with a vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units, number average molecular weight of 68,000) at a concentration of 100 ppm and an ethanol concentration of 500 ppm, pure water was used, and the same treatment as in Example 1 was performed. This was used as Comparative Example 1. The results are shown in Table 1. The separation ratio was 1.9, which meets the preferred range, but the antibody adsorption amount was 100 mg / m³. 2 The above does not meet the preferred range, and it can be said that Comparative Example 1 is not sufficiently effective as a material for adsorbing and removing HCP as an impurity from the culture supernatant of antibody-producing cells.

[0196] (Comparative Example 2)

[0197] Instead of the aqueous solution prepared in Example 1 with a vinylpyrrolidone / vinyl propionate random copolymer (40% molar fraction of vinyl propionate units, number average molecular weight of 68,000) at a concentration of 100 ppm and an ethanol concentration of 500 ppm, an aqueous solution containing polyethyleneimine (molecular weight of 750,000) at a concentration of 100 ppm was used. All other than this, the same treatment as in Example 1 was performed. This was designated as Comparative Example 2. The results are shown in Table 1.

[0198] [Table 1]

[0199]

[0200] Industrial availability

[0201] The porous adsorption material of the present invention can suppress mesh clogging caused by proteins that are not the target of adsorption, and adsorb the target protein. Therefore, it is suitable for materials used to separate and purify proteins that are the target substances.

Claims

1. A porous adsorbent material for adsorbing and removing impurities from a biopharmaceutical raw material solution, wherein at least one surface of the porous adsorbent material has a layer comprising a nonionic polymer, wherein the thickness of the nonionic polymer-containing layer, as determined by time-of-flight secondary ion mass spectrometry (TOF-SIMS), is 1.5 μm or more and 5 μm or less. <Determination of Layer Thickness 2> Based on the obtained mass m / z spectrum, a spectral profile is constructed for any part of the cross section of the porous adsorbent material. Focusing on the characteristic ion species of the nonionic polymer, a line segment is drawn from the flat part of the profile. The distance between the two points where the line segment intersects with the two ends of the peak originating from the nonionic polymer is determined as the thickness of the layer containing the nonionic polymer.

2. The porous adsorbent material according to claim 1, wherein, The inclusions are proteins, and the nonionic polymer is a polymer that inhibits the adhesion of biological components. When performing compositional analysis using time-of-flight secondary ion mass spectrometry (TOF-SIMS), the thickness of the layer containing the polymer that inhibits the adhesion of biological components, determined using the following method, is less than 3 μm. <Determination of Layer Thickness 1> Based on the obtained mass m / z spectrum, spectral profiles are constructed for any three locations on the cross-section of the porous adsorbent material, and the thickness of the layer containing the polymer that inhibits the adhesion of biological components is determined by averaging the values ​​of the three locations.

3. The porous adsorbent material according to claim 1 or 2, wherein, The porous adsorbent material, measured using differential scanning calorimetry (DSC), has an average pore radius of 1 nm or more and 100 nm or less, and a specific surface area of ​​0.05 m². 2 / g or more and 0.5m 2 / g or less.

4. The porous adsorbent material according to claim 1 or 2, wherein, The porous adsorbent material is fibrous.

5. The porous adsorbent material according to claim 1 or 2, wherein, The nonionic polymer is a copolymer polymer having hydrophobic and hydrophilic units.

6. The porous adsorbent material according to claim 5, wherein, The hydrophobic unit is a monocarboxylic acid vinyl ester unit.

7. The porous adsorbent material according to claim 6, wherein, The nonionic polymer also has vinylpyrrolidone units.

8. The porous adsorbent material according to claim 6, wherein, The monocarboxylic acid ethylene ester unit is "-CH(OCO-R)-CH2-", where R is an aliphatic hydrocarbon group with 2 to 5 carbon atoms.

9. The porous adsorbent material according to claim 1 or 2, wherein, The porous adsorbent material selectively adsorbs HCP from a solution containing antibodies and host-derived proteins (HCPs).

10. The porous adsorbent material according to claim 9, wherein, The separation ratio of antibody to HCP is above 1.

5.

11. A separation column for refining biopharmaceuticals, wherein the column contains the porous adsorption material as described in any one of claims 1 to 10.

12. A method for manufacturing biological drugs, comprising: The process of preparing biological pharmaceutical raw material solutions; The process of contacting the biopharmaceutical raw material solution with the porous adsorbent material according to any one of claims 1 to 10.

13. The method for manufacturing a biological drug according to claim 12, characterized in that, A porous hollow fiber membrane and the porous adsorbent material according to any one of claims 1 to 10 are continuously configured to separate / remove inclusions.

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