Methods for synthesizing adsorbents, purifying antibodies, and manufacturing antibodies.

Abstract

A synthetic adsorbent, determined by the following method, exhibits a maximum differential pore volume (mL / g) exceeding 0.05 mL / g under pressure conditions of 0.5 psia to 30.0 psia. <Method for determining differential pore volume (mL / g)> 1. A sample container containing the dried synthetic adsorbent is placed under reduced pressure to below 10 Pa. Mercury, as specified in JIS K8572, is then degassed and filled into the sample container at a pressure of 0.5 psia. 2. For the sample container filled with mercury, the pressure is progressively increased from 0.5 psia to 30.0 psia, and the amount of mercury injected is measured. 3. The differential pore volume (mL / g) is calculated by dividing the increase in mercury injection volume calculated based on the mercury injection volume measured in step 2 by the amount of synthetic adsorbent measured.

Description

[Technical Field]

[0001] This invention relates to a synthetic adsorbent, a method for purifying antibodies, and a method for manufacturing antibodies. [Previous Technology]

[0002] In recent years, the biopharmaceutical industry has developed rapidly due to advancements in gene recombination technology. Among them, antibody pharmaceuticals are seeing increasing production volumes year by year. Antibody pharmaceuticals are very expensive compared to other low-molecular-weight pharmaceuticals. One reason for their high price is the enormous cost of antibody purification.

[0003] Antibody-like proteins are generally produced by culturing recombinant cells with inserted vectors containing genes encoding the target protein. In addition to the target protein, the culture medium contains various components from different culture media, components from the host cell, protein byproducts, and other impurities. Therefore, it is necessary to separate and remove these impurities to the purity required for pharmaceutical production and to purify the target protein.

[0004] After the antibody is broken down by the proteolytic enzyme papain, specific sites of the antibody are cleaved, resulting in regions called Fab and Fc regions. Protein A is an example of a protein that can specifically bind to the Fc region. A method that utilizes the specificity of this protein to adsorb only the antibody and separate it from impurities is the use of affinity chromatography with protein A. This separation method can selectively capture only the target antibody from a culture medium containing a wide variety of impurities. Therefore, it can be used for the purification of most antibody pharmaceuticals.

[0005] Patent Document 1 discloses a purification method for antibodies using affinity chromatography. On the other hand, Patent Document 2 discloses a purification method using activated carbon without affinity chromatography.

[0006] Patent Document 1: Japanese Patent Publication No. 5-504579; Patent Document 2: International Publication No. 2014 / 024514

[0007] Compared to separating agents that utilize general ion exchange or hydrophobic interactions, the affinity separating agent disclosed in Patent Document 1, which uses affinity chromatography with proteins such as protein A, is very expensive. Therefore, antibody pharmaceuticals produced using large quantities of affinity separating agents also become expensive.

[0008] As disclosed in Patent Document 2, the manufacturing cost of antibodies can also be reduced by using a purification method that utilizes activated carbon instead of affinity chromatography. However, in this method, the recovery rate of the target antibody is significantly lower compared to affinity chromatography.

[0009] As mentioned above, it is difficult to achieve low cost and high production in previous antibody purification methods. [Summary of the Invention]

[0010] The object of the present invention is to provide a synthetic adsorbent that can reduce the manufacturing cost of antibodies and efficiently produce antibodies on an industrial scale. Furthermore, the object of the present invention is to provide a method for purifying and manufacturing antibodies, which can reduce the manufacturing cost of antibodies and efficiently produce antibodies on an industrial scale.

[0011] The inventors discovered that by using a synthetic adsorbent with a maximum differential pore volume (mL / g) exceeding a specified value for antibody purification, a low-cost and high-yield antibody purification method can be achieved, thus completing the present invention.

[0012] That is, the gist of the present invention is as follows.

[0013] [1] A synthetic adsorbent, the maximum value of the differential pore volume (mL / g) under pressure conditions of 0.5 pounds per square inch absolute (psia) to 30.0 psia is greater than 0.05 mL / g, as determined by the following method: <Method for determining differential pore volume (mL / g)> 1. The sample container containing the dried synthetic adsorbent is depressurized to below 10 Pa, and the mercury specified in Japanese Industrial Standards (JIS) K8572 is depressurized to below 10 Pa and degassed, and then filled into the sample container at a pressure of 0.5 psia; 2. For the sample container filled with the mercury, the pressure is gradually increased from 0.5 psia to 30.0 psia, and the amount of mercury injected at this time is measured. 3. The differential pore volume (mL / g) is calculated by dividing the increase in mercury injection volume when the pressure is increased by one stage, calculated based on the mercury injection volume determined in step 2, by the determined synthetic adsorbent dose. [2] The synthetic adsorbent as described in [1] is a synthetic adsorbent for antibody purification. [3] The synthetic adsorbent as described in [1] or [2] has pores. [4] The synthetic adsorbent as described in [3] has a pore radius of 3 nm to 15 nm. [5] The synthetic adsorbent as described in any one of [1] to [4] is hydrophobic. [6] The synthetic adsorbent as described in any one of [1] to [5] contains at least one selected from styrene-based resins and acrylic resins. [7] The synthetic adsorbent as described in any one of [1] to [6] has a volume average particle size of 1 μm to 500 μm. [8] The synthetic adsorbent as described in any one of [1] to [7], wherein the value of the corrected roundness obtained by the following method exceeds 0.10; <Method for determining and calculating the value of the corrected roundness> 1. Take a picture of the synthetic adsorbent using an optical microscope; 2. Create an approximate circle of the photographed synthetic adsorbent using the image analysis software WinROOF2018; 3. Clamp the approximate circle with two concentric geometric circles (two concentric circles) according to JIS B0621; 4. Calculate the difference between the radius of the outer circumference circle and the radius of the inner circumference circle in the two concentric circles when the interval between the two concentric circles in 3 is the smallest, and take the roundness as the value; 5. Divide the calculated roundness by the radius of the approximate circle; 6. Perform the determination and calculation of 1 to 5 on more than 100 points, and take the average value as the corrected roundness. [9] The synthetic adsorbent as described in any one of [1] to [8], wherein the crushing strength is 100 gf / particle to 2000 gf / particle.

[10] The synthetic adsorbent as described in any one of [2] to [9], wherein the antibody is a monoclonal antibody.

[11] The synthetic adsorbent as described in

[10] , wherein the monoclonal antibody has a molecular weight of 100,000 or more.

[12] The synthetic adsorbent as described in

[10] or

[11] , wherein the monoclonal antibody is immunoglobulin G.

[13] A method for purifying an antibody, wherein a mixture comprising an antibody and impurities is mixed with the synthetic adsorbent as described in any one of [1] to

[12] and then filtered.

[14] A method for purifying an antibody, comprising: passing a mixture comprising an antibody and impurities through the synthetic adsorbent as described in any one of [1] to

[12] , and recovering a non-adsorbed fraction containing an antibody that was not adsorbed by the synthetic adsorbent.

[15] A method for purifying an antibody, comprising the following steps (i) and (ii): Step (i): a contacting step, wherein a mixed solution containing an antibody and impurities is contacted with a synthetic adsorbent as described in any one of [1] to

[12] , and Step (ii): a separation step, wherein, after step (i), the mixed solution is separated from the synthetic adsorbent.

[16] The method for purifying an antibody as described in

[15] , wherein the preceding part of step (i) comprises the following step (A): Step (A): an ion exchange resin contacting step, wherein a mixed solution containing an antibody and impurities is contacted with an anion exchange resin and / or a cation exchange resin.

[17] The method for purifying an antibody as described in any one of

[13] to

[16] , wherein the impurities comprise substances with a molecular weight of 50,000 or less.

[18] The method for purifying an antibody as described in any one of

[13] to

[17] , wherein the impurities comprise at least one selected from the group consisting of proteins derived from host cells, polymers derived from antibodies, degradation products derived from antibodies, and nucleic acids.

[19] A method for purifying an antibody as described in any one of

[13] to

[18] , wherein the mixture is mixed with a synthetic adsorbent in a liquid with a pH of 3 to 8, the mixture is passed through the synthetic adsorbent, or the mixed solution is contacted with the synthetic adsorbent.

[20] A method for manufacturing an antibody, comprising a method for purifying an antibody as described in any one of

[13] to

[19] .

[0014] Furthermore, the second aspect of the present invention is as follows: <1> A synthetic adsorbent, a fragmented synthetic adsorbent for antibody purification. <2> The synthetic adsorbent as described in <1> has fine pores. <3> The synthetic adsorbent as described in <2>, wherein the radius of the fine pores is 3 nm to 15 nm. <4> The synthetic adsorbent as described in any one of <1> to <3> is hydrophobic. <5> The synthetic adsorbent as described in any one of <1> to <4> comprises at least one selected from styrene-based resins and acrylic resins. <6> The synthetic adsorbent as described in any one of <1> to <5>, wherein the volume average particle size is 1 μm to 500 μm. <7> The synthetic adsorbent as described in any one of <1> to <6>, wherein the antibody is a monoclonal antibody. <8> The synthetic adsorbent as described in <7>, wherein the molecular weight of the monoclonal antibody is 100,000 or more. <9> The synthetic adsorbent as described in <7> or <8>, wherein the monoclonal antibody is immunoglobulin G. <10> A method for purifying an antibody, wherein a mixture comprising antibody and impurities is mixed with a synthetic adsorbent as described in any one of <1> to <9> and then filtered. <11> A method for purifying an antibody, comprising: passing a mixture comprising antibody and impurities through a synthetic adsorbent as described in any one of <1> to <9>, and recovering a non-adsorbed fraction containing antibody that was not adsorbed by the synthetic adsorbent. <12> A method for purifying an antibody as described in <10> or <11>, wherein the impurities comprise substances with a molecular weight of 50,000 or less. <13> A method for purifying an antibody as described in any one of <10> to <12>, wherein the impurities comprise at least one selected from the group consisting of proteins derived from host cells, polymers derived from antibodies, degradation products derived from antibodies, and nucleic acids. <14> A method for purifying an antibody as described in any one of <10> to <13>, wherein the mixing of the mixture with the synthetic adsorbent is carried out in a liquid with a pH of 3 to 8. <15> A method for manufacturing an antibody, comprising the purification method of the antibody as described in any one of <10> to <14>. [Effects of the Invention]

[0015] The synthetic adsorbent of the present invention can reduce the purification and manufacturing costs of antibodies and can also efficiently produce antibodies on an industrial scale.

[0016] The antibody purification method and antibody manufacturing method of the present invention can reduce the purification and manufacturing costs of antibodies and can also efficiently produce antibodies on an industrial scale.

Implementation Method

[0018] The present invention will now be described in detail. The present invention is not limited to the embodiments described below, and various modifications may be made within its scope. Where the expression "~" is used in this specification, it is used to indicate the numerical or physical property values ​​preceding or following it. In this specification, "(meth)acrylic acid" refers to "acrylic acid," "methacrylic acid," or both. "(meth)acrylate" refers to "acrylate," "methacrylate," or both. The same applies to "(meth)acrylic acid".

[0019] (Synthetic Adsorbent) The synthetic adsorbent of the present invention is characterized in that the maximum value of the differential pore volume (mL / g) under pressure conditions of 0.5 psia to 30.0 psia, as determined by the following method, exceeds 0.05 mL / g. <Method for determining differential pore volume (mL / g)> 1. The sample container containing the dried synthetic adsorbent is depressurized to below 10 Pa, and the mercury specified in JIS K8572 is depressurized to below 10 Pa and degassed, and then filled into the sample container at a pressure of 0.5 psia. 2. For the sample container filled with said mercury, the pressure is gradually increased from 0.5 psia to 30.0 psia, and the amount of mercury injected at this time is measured. 3. Calculate the differential pore volume (mL / g), which is obtained by dividing the increase in mercury injection volume when the pressure is increased by one stage, calculated based on the mercury injection volume determined in step 2, by the measured synthetic adsorption dose.

[0020] The mercury infiltration method is a method for determining the volume of pores or micropores. It utilizes the fact that mercury, due to its high surface tension, cannot enter pores or micropores under atmospheric pressure alone. The method involves forcing mercury in using pressure. During the measurement, the pressure is increased in stages, and the amount of mercury injected is detected using a detector such as an electrostatic capacitance type. The increased mercury injection volume is used to calculate the micropore volume. The differential micropore volume (mL / g) is obtained by dividing the increase in mercury injection volume (calculated based on the measured mercury injection volume) by the measured synthetic adsorption dose.

[0021] In the determination of differential pore volume (mL / g), although the magnitude of the pressure increase in each measurement when the pressure increases in stages also depends on the specifications of the pore volume measuring device used, in order to accurately grasp the differential pore volume (mL / g) of the present invention, it is preferably in the range of 2 psia to 10 psia, especially 2 psia to 5 psia.

[0022] In this invention, regarding the differential pore volume (mL / g), specifically as described in the embodiments disclosed below, a pore distribution measuring device (model name "AutoPore IV 9520", manufactured by Micromeritics Instruments Corporation) is used. The differential pore volume (mL / g) is determined by mercury injection method based on the increase in mercury injection amount as the measuring pressure is progressively increased. As an example, the measuring pressure value for the mercury injection amount is preferably set as follows: First measuring pressure value: 9.0 ± 0.5 psia; Second measuring pressure value: 11.0 ± 0.5 psia; Third measuring pressure value: 15.0 ± 0.5 psia; Fourth measuring pressure value: 20.0 ± 0.5 psia; Fifth measuring pressure value: 25.0 ± 0.5 psia.

[0023] The synthetic adsorbent of the present invention is characterized in that the maximum value of the differential pore volume (mL / g) at a test pressure ranging from 0.5 psia to 30.0 psia exceeds 0.05 mL / g. A synthetic adsorbent with a maximum differential pore volume (mL / g) exceeding 0.05 mL / g is one that has fine pores on the surface of the synthetic adsorbent particles with excellent impurity removal properties, resulting in excellent impurity removal and antibody purification efficiency. Because of its excellent impurity removal properties, the maximum value of the differential pore volume (mL / g) of the synthetic adsorbent of the present invention exceeds 0.05 mL / g, preferably 0.1 mL / g or more, more preferably 0.2 mL / g or more, and even more preferably 0.3 mL / g or more. On the other hand, there is no particular limitation on the upper limit of the maximum differential pore volume (mL / g), but from the viewpoint of measurement accuracy or the physical strength of the synthetic adsorbent, it is generally 1.0 mL / g or less.

[0024] A synthetic adsorbent with a maximum differential pore volume (mL / g) exceeding 0.05 mL / g can be obtained, for example, by pulverizing commercially available synthetic adsorbent (synthetic resin) particles or manufactured synthetic adsorbent (synthetic resin) particles as described below to obtain a synthetic adsorbent that satisfies this maximum differential pore volume (mL / g).

[0025] A synthetic adsorbent is a synthetic substance that has optimized the parent structure, micropore structure, surface area, surface polarity, etc., and adsorbs specific organic compounds from solution through physical or chemical interactions.

[0026] In this invention, the synthetic adsorbent refers to an organic synthetic adsorbent. Examples of materials used as synthetic adsorbents include styrene-based resins, acrylic resins, phenolic resins, and amide-based resins. These synthetic adsorbent materials can be used alone or in combination of two or more. Among these synthetic adsorbent materials, styrene-based resins and acrylic resins are preferred in terms of low cost and excellent hydrophobicity, and styrene-based resins are more preferred.

[0027] Hydrophobicity refers to the nonpolar property of water molecules, which has a low affinity for water molecules, based on their ability to aggregate in aqueous solutions. Interactions utilizing this attraction are called hydrophobic interactions. Examples of hydrophobic functional groups include those of alkyl, phenyl, and benzyl hydrocarbons.

[0028] In this specification, a styrene-based resin is one in which at least 50% by mass of the structural units derived from aromatic vinyl monomers constitute 100% of all monomeric units constituting the styrene-based resin. This proportion is preferably 80% by mass or more, which is advantageous in terms of the amount of impurities adsorbed into the synthetic adsorbent, the physical strength of the synthetic adsorbent, and the ability to form fine pores. The styrene-based resin may also contain structural units derived from sources other than aromatic vinyl monomers.

[0029] Examples of aromatic vinyl monomers include: styrene, methylstyrene, ethylstyrene, α-methylstyrene, chlorostyrene, chloromethylstyrene, bromobutylstyrene, and other aromatic vinyl monomers; and crosslinked aromatic vinyl monomers such as divinylbenzene, bis(vinylphenyl)ethane, divinylnaphthalene, and 2,4,6-trivinylethylbenzene. These aromatic vinyl monomers can be used alone or in combination with two or more. Among these aromatic vinyl monomers, it is preferable to include a crosslinked aromatic vinyl monomer in terms of the amount of impurities adsorbed onto the synthetic adsorbent, the physical strength of the synthetic adsorbent, and the pore-forming properties; more preferably, it is preferable to use both an aromatic vinyl monomer and a crosslinked aromatic vinyl monomer; and even more preferably, it is preferable to use both styrene and divinylbenzene.

[0030] Of the 100% by mass of all monomeric units constituting the styrene-based resin, the content of structural units derived from aromatic monovinyl monomers in the styrene-based resin is preferably 0% to 99% by mass, more preferably 20% to 80% by mass. When the content of structural units derived from aromatic monovinyl monomers is above the lower limit, the synthetic adsorbent exhibits excellent flexibility. When the content of structural units derived from aromatic monovinyl monomers is below the upper limit, the dissolution, swelling, or shrinkage of the synthetic adsorbent in the solvent can be suppressed.

[0031] Of the 100% by mass of all monomeric units constituting the styrene-based resin, the content of structural units derived from cross-linked aromatic vinyl monomers in the styrene-based resin is preferably 1% to 100% by mass, more preferably 20% to 80% by mass. When the content of structural units derived from cross-linked aromatic vinyl monomers is above the lower limit, the dissolution, swelling, or shrinkage of the synthetic adsorbent in the solvent can be suppressed. When the content of structural units derived from cross-linked aromatic vinyl monomers is below the upper limit, the synthetic adsorbent exhibits excellent flexibility.

[0032] In terms of the excellent hydrophobicity of the synthetic adsorbent, the content of structural units derived from other monomers other than aromatic monovinyl monomers and crosslinked aromatic vinyl monomers in the styrene-based resin is preferably 10% by mass or less, more preferably 1% by mass or less, and even more preferably 0% by mass or less, out of 100% by mass of all monomer units constituting the styrene-based resin.

[0033] In this specification, an acrylic resin is one in which at least 50% by mass of structural units derived from (meth)acrylates constitute 100% of all monomeric units constituting the acrylic resin. For the purpose of excellent antibody recovery, this proportion is preferably 80% by mass or more. Acrylic resins may also contain structural units derived from sources other than (meth)acrylates.

[0034] Examples of (meth)acrylates include: methyl methacrylate, ethyl methacrylate, butyl methacrylate, stearyl methacrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate, and other alkyl methacrylates; hydroxyl-containing (meth)acrylates such as hydroxyl ethyl methacrylate, hydroxypropyl methacrylate, and glyceryl mono(meth)acrylate; glycidyl methacrylate, 4,5-epoxybutyl methacrylate, and 9,10-epoxybutyl methacrylate. The methacrylates include epoxy-containing esters such as aliphatic esters; methacrylamide derivatives such as (meth)acrylamide, dimethacrylamide, and hydroxyethyl (meth)acrylamide; cyano-containing methacrylates such as (meth)acrylonitrile; alkyl dimethacrylates such as ethylene glycol dimethacrylate; polyalkylene glycol dimethacrylates such as polyethylene glycol dimethacrylate; and crosslinked methacrylates such as N,N'-alkylbis(meth)acrylamide, glyceryl dimethacrylate, and trimethylolpropane trimethacrylate. These methacrylates can be used alone or in combination with two or more. Among these methacrylates, those containing crosslinked methacrylates are preferred in terms of ease of functionalization and excellent physical strength of the synthesized adsorbent, and more preferably include ethylene glycol dimethacrylate.

[0035] Of all 100% by mass of the monomeric units constituting the acrylic resin, the content of crosslinked (meth)acrylate units in the acrylic resin is preferably 1% to 100% by mass, more preferably 20% to 80% by mass. When the content of crosslinked (meth)acrylate units is above the lower limit, the dissolution, swelling, or shrinkage of the synthesized adsorbent in the solvent can be suppressed. When the content of crosslinked (meth)acrylate units is below the upper limit, the synthesized adsorbent exhibits excellent flexibility.

[0036] The synthetic adsorbent of the present invention is obtained by crushing synthetic resin particles or commercially available products manufactured by known polymerization methods such as suspension polymerization or emulsion polymerization.

[0037] Crushing refers to the process of reducing the size of the synthetic adsorbent through physical force. There are no particular limitations on the apparatus used for crushing, as long as it can reduce the size of the synthetic adsorbent through physical force; examples include mortars, mixers, ball mills, hammer mills, and needle mills. Among these crushing apparatuses, a ball mill is preferred in terms of its ability to finely pulverize the synthetic adsorbent and easily control the particle size distribution.

[0038] In terms of efficiently increasing specific surface area and excellent impurity removal, the synthetic adsorbent of the present invention preferably has fine pores.

[0039] Regarding the formation of fine pores in the synthetic adsorbent, a pore-forming agent is simply used during polymerization when the synthetic resin is obtained.

[0040] The radius of the pores in the synthetic adsorbent is preferably 3 nm to 15 nm, more preferably 5 nm to 10 nm. When the radius of the pores in the synthetic adsorbent is above the lower limit, the diffusion of impurities into the synthetic adsorbent is excellent, and the removal of impurities is excellent. When the radius of the pores in the synthetic adsorbent is below the upper limit, the adsorption amount of impurities into the synthetic adsorbent is excellent, and the decrease in yield caused by the diffusion of antibodies into the synthetic adsorbent can be suppressed. That is, the antibody recovery rate and antibody productivity during antibody purification are excellent.

[0041] The radius of the pores of the synthetic adsorbent is set as the mode radius of the pores measured using nitrogen and a pore distribution measuring device.

[0042] The radius of the pores of the synthetic adsorbent can be adjusted by controlling the type or amount of the porosifectant and the polymerization rate when the synthetic resin is obtained.

[0043] The volume average particle size of the synthetic adsorbent is preferably 1 μm to 500 μm, more preferably 10 μm to 200 μm. When the volume average particle size of the synthetic adsorbent is above the lower limit, the filterability during antibody purification is excellent, and the antibody recovery rate and antibody productivity are excellent. When the volume average particle size of the synthetic adsorbent is below the upper limit, the surface area of ​​the synthetic adsorbent can be increased, and the impurity removal performance is excellent.

[0044] The volume average particle size of the synthetic adsorbent was set as the value obtained by measuring it using a laser diffraction-scattering particle size analyzer. The sample used was obtained by immersing the synthetic adsorbent in a 20% (v / v) aqueous ethanol solution and removing adhering moisture by filtration.

[0045] The corrected roundness value of the synthetic adsorbent of the present invention is preferably greater than 0.10, more preferably greater than 0.40. When the corrected roundness value is greater than 0.10, the synthetic adsorbent has high diffusivity into the micropores and excellent impurity removal performance. There is no particular upper limit to the corrected roundness, but it is usually below 1.00.

[0046] Here, the term "roundness" is defined in JIS B0621 as "the magnitude of the deviation between a circular shape and a geometrically perfect circle." Roundness is "the difference in radii between the two concentric geometric circles when the distance between them is minimized when the object (circular shape) is held between them." The "corrected roundness" of this invention is obtained by dividing this roundness by the radius of the approximate circle of the synthetic adsorbent photographed when the roundness was calculated. It is equivalent to correcting the change in roundness value caused by particle size. A roundness closer to zero indicates a closer approximation to a geometric circle, while a larger roundness indicates a greater deviation from a geometric circle.

[0047] The corrected roundness of the present invention is obtained using the following method. Furthermore, the unit of length for the following roundness and the radius of the approximate circle is the same. <Method for determining and calculating the corrected roundness> 1. Photograph the synthetic adsorbent using an optical microscope. 2. Create an approximate circle of the photographed synthetic adsorbent using image analysis software WinROOF2018. 3. Clamp the approximate circle using two concentric geometric circles (two concentric circles) concentric with it, according to JIS B0621. 4. Calculate the difference between the radius of the outer circumference circle and the radius of the inner circumference circle of the two concentric circles (where the interval between the two concentric circles in step 3 is minimized) as the roundness. 5. Divide the calculated roundness by the radius of the approximate circle. 6. Perform the measurements and calculations of steps 1 to 5 on at least 100 points, and use the average value as the corrected roundness.

[0048] The crushing strength (physical strength) of the synthetic adsorbent is preferably 100 gf / particle to 2000 gf / particle, more preferably 300 gf / particle to 1000 gf / particle. Generally, the higher the crushing strength, the less likely it is to break during use, and therefore it is preferred. However, synthetic adsorbents with excessively high crushing strength cannot withstand the swelling and shrinkage caused by changes in the composition of the solvent during liquid circulation, and thus break. The range of the aforementioned crushing strength is preferred in terms of a balance between strong loads caused by the stacking of synthetic adsorbent particles and physical impacts such as collisions caused by stirring, and the ability to withstand swelling and shrinkage caused by changes in the composition of the solvent.

[0049] Here, crush strength is the average value of the force required to cause an object to lose its original shape (become destroyed) when subjected to crushing (compression) force. Specifically, crush strength is measured using the method described in the embodiments disclosed later. In the embodiments disclosed later, the crush strength was measured on the synthetic adsorbent before it was crushed. This is because the particles in the crushed synthetic adsorbent are too small to obtain a measurement result. Crushing strength is the strength of a material, and generally, for the same composition, the smaller the value, the higher the strength. Therefore, it is assumed that the value will not decrease after crushing, and the measured value of the crushing strength of the synthetic adsorbent before crushing is greater than or equal to the measured value of the synthetic adsorbent after crushing.

[0050] (Antibody) The synthetic adsorbent of the present invention is preferably used for the purification of antibodies.

[0051] Antibodies are proteins produced in the body as a result of antigen stimulation through an immune response, and are those that have the activity of specifically binding to antigens.

[0052] Examples of antibodies include: mouse antibodies, alpaca antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibodies that have altered the Fc region of these antibodies, antibodies that have undergone multivalent modification of these antibodies, and antibodies that have altered a portion of these antibodies. Examples of antibody molecular types include: immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), Fab, Fc, Fc-fusion protein, VH, VL, VHH, Fab'2, scFv, scFab, scDb, scDbFc, etc. Among these antibodies, those with superior singularity are preferably antibodies derived from the same cell, homologous antibodies, and more preferably monoclonal antibodies.

[0053] Monoclonal antibodies are antibodies produced by monoclonal antibody-producing cells. They have a primary structure, meaning their amino acid sequences are uniform. Therefore, the class, subclass, allotype, L-chain, etc., are also uniform.

[0054] Examples of monoclonal antibodies include immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin D (IgD), and immunoglobulin E (IgE). Among these monoclonal antibodies, IgG, IgD, and IgE are preferred in terms of their resistance to adsorption by synthetic adsorbents, with IgG being more preferred.

[0055] The molecular weight of the monoclonal antibody is preferably 100,000 to 9,000,000, more preferably 300,000 to 2,000,000. When the molecular weight of the monoclonal antibody is above the lower limit, the yield decrease caused by the diffusion of the antibody into the synthetic adsorbent can be suppressed. Furthermore, when the molecular weight of the monoclonal antibody is below the upper limit, the monoclonal antibody is less likely to precipitate in water and is easily soluble.

[0056] The molecular weight of the monoclonal antibody is set to the value obtained by size exclusion chromatography using the molecular sieve effect.

[0057] (Method for purifying antibodies) There are no particular limitations on the method of purifying antibodies using the synthetic adsorbent of the present invention, for example, the following methods (1) to (3) can be listed.

[0058] (1) A method for purifying an antibody, wherein a mixture comprising an antibody and impurities is mixed with the synthetic adsorbent of the present invention and then filtered. That is, the synthetic adsorbent containing impurities is separated from the antibody dissolved in the liquid by filtration.

[0059] (2) A purification method in a so-called flowthrough mode, comprising: passing a mixture containing antibodies and impurities through the synthetic adsorbent of the present invention, and recovering a non-adsorbed fraction containing antibodies that was not adsorbed by the adsorbent.

[0060] (3) A method for purifying an antibody, comprising the following step (A) preceding steps (i) and (ii). Step (i): a contacting step, in which a mixed solution containing antibodies and impurities is contacted with the synthetic adsorbent of the present invention. Step (ii): a separation step, in which the mixed solution is separated from the synthetic adsorbent after step (i). Step (A): an ion exchange resin contacting step, in which the mixed solution containing antibodies and impurities is contacted with anion exchange resin and / or cation exchange resin. By utilizing the desalting treatment performed in step (A), the impurity removal efficiency, antibody purification efficiency, and antibody recovery efficiency of the synthetic adsorbent of the present invention can be improved. Furthermore, a mixture of anion exchange resin and / or cation exchange resin used in step (A) may also be mixed with the synthetic adsorbent used in step (i). Step (i) may be performed in a batch manner or in a flow-through manner; in the case of a flow-through manner, step (ii) may be omitted.

[0061] Impurities are those that need to be removed for the purpose of using antibodies as pharmaceuticals. Examples include culture medium components needed for culturing recombinant cells, proteins and metabolites from which antibodies derived from cells are removed, and nucleic acids. Specifically, impurities include at least one selected from the group consisting of proteins derived from host cells, polymers derived from antibodies, degradation products derived from antibodies, and nucleic acids.

[0062] Proteins derived from host cells are proteins produced during the growth process of cells that produce antibodies, cell fragments of dead cells, etc. Examples include modified proteins that have undergone removal, oxidation, or deaminement of glycan components, and enzymes dissolved from host cells.

[0063] Examples of enzymes that can be lysed from host cells include, for example, desaccharidases, proteolytic enzymes, oxidoreductases, and amino acid isomerases. Examples of desaccharidases include, for example, sialidase, galactosidase, and glycanase. Examples of proteolytic enzymes include, for example, serine protease, esterase, cysteine ​​protease, trypsin-like protease, aminopeptidase, aspartate protease, and cathepsin. Examples of oxidoreductases include, for example, thioredoxin reductase and other thioredoxin-related enzymes.

[0064] So-called antibody-derived polymers are polymers formed by the polymerization of antibodies under conditions such as heat, catalyst, and pH. Examples include heat-modified polymers of human gamma globulin and polymers generated by oxidation.

[0065] The so-called antibody decomposition products are fragments of antibodies that are broken down by protein-degrading enzymes, heat, pH conditions, etc., causing the peptide bonds or disulfide bonds of the antibodies to break.

[0066] A nucleic acid is a long-chain polynucleotide composed of nucleotides containing purine, pyrimidine bases, pentose sugars and phosphates as basic units, with the phosphates bonded between the nucleotides via diester bonds between the 3' and 5' carbons of the sugar. Examples include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their breakdown products.

[0067] The molecular weight of the impurity is preferably 1,000 to 50,000, more preferably 5,000 to 25,000. When the molecular weight of the impurity is above the lower limit, the impurity has a hydrophobic portion and is easily adsorbed by the synthetic adsorbent. In addition, when the molecular weight of the impurity is below the upper limit, the impurity has excellent diffusivity into the synthetic adsorbent.

[0068] The molecular weight of the impurities is set to a value obtained by size exclusion chromatography using the molecular sieve effect.

[0069] The filtration in the method described in (1) is carried out by separating the solid and liquid using a filter material.

[0070] Examples of filter materials include: hydrophilic styrene-based polymer membranes, hydrophilic aromatic ether-based polymer membranes, hydrophilic fluorine-based polymer membranes, hydrophilic olefin-based polymer membranes, cellulose-based membranes, (meth)acrylic acid-based polymer membranes, (meth)acrylonitrile-based polymer membranes, and vinyl alcohol-based polymer membranes. Among these filter materials, hydrophilic fluorine-based polymer membranes, hydrophilic styrene-based polymer membranes, and cellulose-based membranes are preferred in terms of excellent hydrophilicity, and hydrophilic fluorine-based polymer membranes are even more preferred.

[0071] The pore size of the filter material is preferably 0.1 μm to 30 μm, more preferably 0.2 μm to 10 μm. When the pore size of the filter material is above the lower limit value, the liquid flow performance of the filtration is excellent. In addition, when the pore size of the filter material is below the upper limit value, the separation performance between solids and liquids is excellent.

[0072] The pore size of the filter material is measured using a scanning electron microscope (SEM) and the obtained images.

[0073] In the method described in (3), as a method of contacting the mixed solution containing antibodies and impurities with the synthetic adsorbent of the present invention in step (i), a method of mixing the mixed solution containing antibodies and impurities with the synthetic adsorbent of the present invention can be listed. Alternatively, as in the method described in (2), a flow-through mode in which the mixed solution is passed through a column filled with the synthetic adsorbent of the present invention can also be used.

[0074] In the method described in (3), the anion exchange resin used in step (A) can be exemplified as: a basic anion exchange resin having quaternary ammonium (trimethylammonium or dimethylethanolamine) groups as exchange groups in a crosslinked polymer containing monovinyl aromatic monomers and crosslinking aromatic monomers, and exhibiting basicity by dissociating in the same manner as strong bases such as sodium hydroxide and potassium hydroxide. In particular, it is preferred to have ion exchange properties throughout the entire pH range (0-14), and it is even more preferred to be a strongly basic anion exchange resin. As commercially available basic anion exchange resins, examples include: the SA series, PA series, and UBA series of Diaion (registered trademark) manufactured by Mitsubishi Chemical Corporation. As a specific resin of basic anion exchange resin, it has quaternary ammonium (trimethylammonium or dimethylethanolamine) groups as exchange groups, and the salt form can be OH type, Cl type, etc., of which the OH type has excellent desalination efficiency and is preferred.

[0075] On the other hand, cation exchange resins are ion exchange resins containing sulfonic acid groups (-SO3H) and other exchange groups in a crosslinked polymer comprising monovinyl aromatic monomers and crosslinkable aromatic monomers. Examples include acidic cation exchange resins that dissociate in the same way as mineral acids such as hydrochloric acid and sulfuric acid to exhibit acidity. Particularly preferred are those exhibiting ion exchange capacity across the entire pH range (0-14), and strongly acidic anion exchange resins are preferred. Examples of commercially available acidic cation exchange resins include, for example, the SK series, PK series, and UBK series of Diaion (registered trademark) manufactured by Mitsubishi Chemical Corporation. Specific resins of acidic cation exchange resins have sulfonic acid groups as exchange groups, and their salt form is generally H-form or Li-form, with H-form being preferred in terms of desalination efficiency.

[0076] Furthermore, when ion exchange resins are broadly classified according to their structural properties, they can be divided into "gel type" and "porous type." However, the acidic cation exchange resin and the basic anion exchange resin used in this invention are preferably both gel type. That is, compared with porous ion exchange resins, gel type ion exchange resins have a larger ion exchange capacity per unit volume and higher physical strength (compressive strength), and therefore can be used for a long time.

[0077] In the desalination process of step (A), only one of the anion exchange resin and the cation exchange resin may be used, but in terms of removing both cationic and anionic impurities, it is preferable to use the anion exchange resin and the cation exchange resin in combination.

[0078] The desalting process in step (A) can be carried out by mixing a mixed solution containing antibodies and impurities with anion exchange resin and / or cation exchange resin, or by passing a mixed solution containing antibodies and impurities into anion exchange resin bed and / or cation exchange resin bed, preferably a mixed bed of anion exchange resin and cation exchange resin.

[0079] In any case, from the viewpoint of improving the efficiency of impurity removal, antibody purification and antibody recovery caused by the desalting treatment, it is preferable that the treatment solution obtained by the desalting treatment in step (A) exhibits a low conductivity of less than 10 mS / cm, especially less than 5 mS / cm, due to the removal of ionic impurities.

[0080] In the methods described in (1) to (3), the mixing of the mixture with the synthetic adsorbent, the passing of the mixture through the synthetic adsorbent, or the contact between the mixed solution and the synthetic adsorbent is preferably carried out in a liquid with a pH of 3 to 8, and more preferably in a liquid with a pH of 4 to 6. When the pH is above the lower limit, the stability of the antibody can be maintained. In addition, when the pH is below the upper limit, the adsorption of the antibody to the synthetic adsorbent can be inhibited. Therefore, acids, bases, etc., can be added to the mixture or mixed solution containing the antibody and impurities as needed to adjust the pH.

[0081] (Antibody manufacturing method) The antibody manufacturing method of the present invention includes the antibody purification method described above.

[0082] In addition to the antibody purification step described above, the antibody manufacturing method of the present invention may also include a cell culture step, a purification step using an ion exchange resin, a purification step using size exclusion chromatography, and a purification step using membrane separation.

[0083] (Applications) The synthetic adsorbent of the present invention can reduce the manufacturing cost of antibodies and efficiently produce antibodies on an industrial scale. Furthermore, the antibody purification method and antibody manufacturing method of the present invention can reduce the manufacturing cost of antibodies and efficiently produce antibodies on an industrial scale. [Examples]

[0084] Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to the description of the following examples as long as it does not depart from its spirit.

[0085] (Determination of volume average particle size and particle size distribution) For synthetic adsorbents (A) to synthetic adsorbents (G), the volume average particle size and particle size distribution were determined using a laser diffraction-scattering particle size analyzer (model name "MLS-2000e", manufactured by Seishin Enterprise Co., Ltd.).

[0086] (Determination of Differential Pore Volume) For synthetic adsorbents (A) to (G), the differential pore volume was measured using a pore distribution measuring device (AutoPore IV 9520, manufactured by Micromeritics Instruments Corporation) by mercury injection method, when the pressure was gradually increased from 0.5 psia to 30.0 psia, and the maximum value was determined. As an example, the actual measured pressure and the measured value of the differential pore volume of synthetic adsorbents (A) to (G) are shown in Table 1 below.

[0087] [Table 1] Synthetic adsorbent (A) Synthetic adsorbent (B) Synthetic adsorbent (C) Synthetic adsorbent (D) Synthetic adsorbent (E) Synthetic adsorbent (F) Synthetic adsorbent (G) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) pressure (psia) Differential pore volume (mL / g) 8.94995 0 8.94995 0 8.94995 0 8.94995 0 8.94995 0 8.94995 0 8.94995 0 8.9742 0.00521 8.9742 0.00495 8.9742 0.00156 8.9728 0.00113 8.9728 0.000996 8.9728 0.000888 8.97447 0.00052 11.1633 0.34807 11.1633 0.38265 11.1633 0.11198 11.1613 0.08695 11.1613 0.07989 11.1613 0.08337 11.1622 0.04394 14.8842 0.21187 14.8842 0.26188 14.8842 0.06854 14.8764 0.10011 14.8764 0.09784 14.8764 0.14054 14.8829 0.02075 19.8646 0.09594 19.8646 0.10969 19.8646 0.0357 19.8642 0.07694 19.8642 0.1093 19.8642 0.13576 19.8654 0.00749 24.8525 0.04183 24.8525 0.04355 24.8525 0.01821 24.8574 0.05143 24.8574 0.1310 24.8574 0.09784 24.8658 0.00576

[0088] (Determination of pore radius) For synthetic adsorbents (D) to (G), the majority radius of the pores was measured using nitrogen gas and a pore distribution measuring device (model name "ASAP2024", manufactured by Micromeritics Instruments Corporation). Furthermore, the pore radius did not change significantly before and after pulverization.

[0089] (Determination and calculation of corrected roundness) Photographs of synthetic adsorbents (A) to (G) were taken using a Nikon SMZ18 (manufactured by Nikon Solutions, Inc.). The radii of the two concentric circles and the radius of the approximate circle were measured using the image analysis software WinROOF2018 (manufactured by Mitani Corporation). Roundness was calculated using the methods described above, as well as the corrected roundness obtained by dividing the roundness by the radius of the approximate circle.

[0090] (Determination of Crushing Strength) Particles of the synthetic adsorbent (A) to (F), before crushing (using the same substance for synthetic adsorbents (A) to (C) and (E), were immersed in demineralized water and stored therejust before the determination. Then, at least 30 particles of the synthetic adsorbent before crushing were randomly selected, and their strength was measured using a Chatilon force measurement CS225 (AMETEK TEST & CALIBRATION INSTRUMENTS). The average strength of all particles was calculated. Furthermore, the synthetic adsorbent (G) has a small particle size (μm), making the determination of its crushing strength difficult.

[0091] (Determination of impurity concentration and antibody recovery rate) The culture medium used for the monoclonal antibody (immunoglobulin G, molecular weight: 150,000) was a culture medium obtained by removing CHO cells after antibody production from Chinese hamster ovary cells (CHO cells) and clarifying the medium. The pH of the culture medium, which was 7.4, was adjusted to 4.5 using a 5 mol / L aqueous acetic acid solution. Subsequently, 0.2 g of each of the synthetic adsorbents (A) to (G) were weighed into 15 mL containers, and 4 mL of the prepared culture medium was added to each container. After addition, the mixture was shaken for 8 hours and then mixed. Then, the synthetic adsorbents were removed by filtration using a membrane filter (Merck, 0.22 μm pore size, polyvinylidene fluoride (PVDF) membrane) to obtain a synthetic adsorbent-treated solution. The concentration of host cell protein (HCP) [ng / mL] was determined by ELISA using a kit (Enzyme-Linked Immunosorbent Assay (ELISA) Kit F550 (CHO Host Cell Protein, 3Rd Generation)). The logarithmic reduction value (LRV) of the HCP concentration was calculated based on the HCP concentration before and after treatment. The antibody recovery rate [%] was determined by high-pressure liquid chromatography (HPLC) using an affinity chromatography column (POROS A20 μm Column, Thermo Fisher Scientific). The solvent was phosphate-buffered saline (pH 7.4), and the eluent was 20 mmol / L phosphate buffer (pH 2.8, 150 mg / L). (mmol / L).

[0092] (Determination of conductivity) The conductivity of the culture supernatant or the desalted solution of the culture supernatant was measured using a HORIBA F-74 benchtop pH meter with conductivity electrode 3552-10D.

[0093] [Example 1] A commercially available synthetic adsorbent (trade name "Sepabeads 825L", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment) was pulverized using a mortar and pestle. The adsorbent was then washed with a 20% (v / v) ethanol aqueous solution using a filter membrane (manufactured by Merck, 5 μm pore size) and filtered to remove adhering moisture, thus obtaining synthetic adsorbent (A). The evaluation results of the obtained synthetic adsorbent (A) are shown in Table 2.

[0094] [Example 2] A commercially available synthetic adsorbent (trade name "Sepabeads 825L", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment) was pulverized using a mortar and pestle, granulated using a 20 μm sieve, washed with a 20% vol% ethanol aqueous solution using a filter membrane (manufactured by Merck, 5 μm pore size), and filtered to remove adhering moisture, thereby obtaining synthetic adsorbent (B). The evaluation results of the obtained synthetic adsorbent (B) are shown in Table 2.

[0095] [Example 3] A commercially available synthetic adsorbent (trade name "Sepabeads 825L", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment) was pulverized using a mortar and pestle, granulated using a 63 μm sieve, washed with a 20% vol% ethanol aqueous solution using a filter membrane (manufactured by Merck, 5 μm pore size), and filtered to remove adhering moisture, thereby obtaining synthetic adsorbent (C). The evaluation results of the obtained synthetic adsorbent (C) are shown in Table 2.

[0096] [Comparative Example 1] The styrene-divinylbenzene copolymer that had undergone porous treatment was not pulverized. Instead, it was washed with a 20% vol% ethanol aqueous solution using a filter membrane (manufactured by Merck, 5 μm pore size), and the adsorbed moisture was removed by filtration to obtain an unpulverized synthetic adsorbent (G). The evaluation results of the obtained synthetic adsorbent (G) are shown in Tables 2 and 3.

[0097] [Table 2] Types of synthetic adsorbents Particle size distribution [μm] Differential pore volume [mL / g] Correcting roundness Crushing strength [gf / grain] HCP concentration [ng / mL] LRV antibody recovery rate [%] d (0.1) d (0.5) d (0.9) Culture medium - - - - - - - 512000 - - Example 1 Synthetic adsorbent (A) 4 27 101 0.348 0.722 316 3950 2.5 78 Example 2 Synthetic adsorbent (B) 38 99 256 0.383 0.764 316 5770 2.2 88 Example 3 Synthetic adsorbent (C) 87 139 221 0.112 0.675 316 32700 1.3 93 Comparative Example 1 Synthetic adsorbent (G) 86 118 162 0.044 0.052 - 374000 0.2 100

[0098] As can also be seen from Table 2, compared with the synthetic adsorbent (G) with a differential pore volume of less than 0.05 mL / g manufactured in Comparative Example 1, the synthetic adsorbents (A) to (C) with a differential pore volume exceeding 0.05 mL / g manufactured in Examples 1 to 3 have superior HCP concentration, LRV, and antibody recovery rate. In particular, the synthetic adsorbent (A) of Example 1 with a small volume average particle size has a low HCP concentration and excellent impurity removal performance. In addition, the synthetic adsorbent (C) of Example 3 with a large volume average particle size has a high antibody recovery rate and can efficiently produce antibodies. Furthermore, the relationship between the measurement pressure and the differential pore volume (increased pore volume) of the synthetic adsorbents (A), (B), (C), and (G) is shown in Figure 1. As shown in Figure 1, the maximum value of the differential pore volume of synthetic adsorbents (A), (B), and (C) is significantly larger than that of synthetic adsorbent (G).

[0099] [Example 4] A commercially available synthetic adsorbent (trade name "Sepabeads 850", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment, with a pore radius of 5 nm) was pulverized using a mortar and pestle. The adsorbent was then washed with a 20% (v / v) aqueous ethanol solution using a filter membrane (manufactured by Merck, with a pore size of 5 μm) and filtered to remove adhering moisture, thus obtaining synthetic adsorbent (D). The evaluation results of the obtained synthetic adsorbent (D) are shown in Table 3.

[0100] [Example 5] A commercially available synthetic adsorbent (trade name "Sepabeads 825L", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment, with a pore radius of 7 nm) was pulverized using a mortar and pestle. The adsorbent was then washed with a 20% (v / v) ethanol aqueous solution using a filter membrane (manufactured by Merck, with a pore size of 5 μm) and filtered to remove adhering moisture, thus obtaining the synthetic adsorbent (E). The evaluation results of the obtained synthetic adsorbent (E) are shown in Table 3.

[0101] [Example 6] A commercially available synthetic adsorbent (trade name "Sepabeads HP21", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment, with a pore radius of 11 nm) was pulverized using a mortar and pestle, washed with a 20% vol% ethanol aqueous solution using a filter membrane (manufactured by Merck, with a pore size of 5 μm), and the adsorbed moisture was removed by filtration to obtain the synthetic adsorbent (F). The evaluation results of the obtained synthetic adsorbent (F) are shown in Table 3.

[0102] [Table 3] Types of synthetic adsorbents Volume average particle size [μm] fine hole radius [nm] Differential pore volume [mL / g] Correcting roundness Crushing strength [gf / grain] HCP concentration [ng / mL] LRV antibody recovery rate [%] Culture medium - - - - - - 512000 - - Example 4 Synthetic adsorbent (D) 72 5 0.100 0.635 546 63500 0.8 96 Example 5 Synthetic adsorbent (E) 55 7 0.131 0.619 316 4100 2.0 91 Example 6 Synthetic adsorbent (F) 112 11 0.141 0.627 391 1700 2.5 62 Comparative Example 1 Synthetic adsorbent (G) 118 7 0.044 0.052 - 374000 0.2 100

[0103] As can also be seen from Table 3, compared with the uncrushed synthetic adsorbent (G) manufactured in Comparative Example 1, the synthetic adsorbents (D) to (F) manufactured in Examples 4 to 6 have superior HCP concentrations, LRVs, and antibody recovery rates. In particular, the synthetic adsorbent (F) of Example 6, with its large pore radius, has a low HCP concentration and excellent impurity removal performance. In addition, the synthetic adsorbent (D) of Example 4, with its small pore radius, has a high antibody recovery rate, enabling efficient antibody production.

[0104] [Example 7] A commercially available synthetic adsorbent (trade name "Sepabeads 825L", manufactured by Mitsubishi Chemical Corporation, a styrene-divinylbenzene copolymer that has undergone porous treatment) was pulverized using a mortar and pestle. After being immersed in a 20% (v / v) aqueous ethanol solution, it was filtered by suction using a filter membrane (manufactured by Merck, 5 μm pore size) to obtain the synthetic adsorbent (H). The maximum differential pore volume of the synthetic adsorbent (H) was 0.131 mL / g. The culture medium for the monoclonal antibody (immunoglobulin G, molecular weight: 150,000) was the supernatant of the culture medium after antibody production by cells derived from Chinese hamster ovaries (CHO cells) followed by removal of the CHO cells and clarification. The pH of the culture medium, which was 7.4, was adjusted to 4.5 using a 5 mol / L aqueous acetic acid solution. Subsequently, 1.5 g of the synthetic adsorbent (H) and 30 mL of the prepared culture medium were measured into a container, shaken at 25°C for 8 hours, and mixed. Then, the synthetic adsorbent was removed by centrifugation and filtration using a membrane (Merck, 0.22 μm pore size) to obtain batches of the synthetic adsorbent treatment solution. The pH of the obtained batches of the synthetic adsorbent treatment solution was adjusted to 3.5 with 1 mol / L hydrochloric acid and allowed to stand at 25°C for 2 hours. Then, the pH was adjusted to 5.0 with 1 mol / L Tris(hydroxymethyl)aminomethane (Tris) solution and filtered to obtain the virus inactivation solution. The obtained virus inactivation solution was diluted 4-fold with MilliQ water and added to an anion exchange chromatography column (trade name "ChromSpeed ​​Q103", manufactured by Mitsubishi Chemical Corporation) equilibrated with 20 mmol / L trihydrochloric acid buffer (pH 8.5). After addition, 5 column volumes of equilibration buffer were bubbled into the column. The non-adsorbed fraction was used as the ChromSpeed ​​Q103 eluent. The obtained ChromSpeed ​​Q103 eluent was adjusted to pH 4.5 with 5 mol / L acetic acid and added to a cation exchange chromatography column (trade name "ChromSpeed ​​S103", manufactured by Mitsubishi Chemical Corporation) equilibrated with 20 mmol / L acetate buffer (pH 4.5). After the addition was completed, the adsorbed fraction was eluted into the eluent (20 mmol / L acetate buffer with 500 mmol / L salt dissolved, pH 4.5), and the eluent was used as the three-stage purified sample.

[0105] The concentrations of HCP and antibodies were determined using the following method for the culture supernatant, the obtained synthetic adsorbent treatment solution, and the three-stage purification solution. The concentration of host cell-derived protein (HCP) [ng / mL] was determined by ELISA using a kit (ELISA kit F550 (CHO Host Cell Protein, 3Rd Generation)). The antibody concentration [HCP / Mab ppm] was determined by high-performance liquid chromatography (HPLC) using an affinity chromatography column (POROS A20 μm Column, Thermo Fisher Scientific). The antibody recovery rate [%] was determined by HPLC using an affinity chromatography column (POROS A20 μm Column, Thermo Fisher Scientific). Phosphate-buffered saline (pH 7.4) was used as the solvent, and 20 mmol / L phosphate buffer (pH 2.8, 150 mg / L saline) was used as the eluent. (mmol / L).

[0106] [Comparative Example 2] The culture medium used for the monoclonal antibody (immunoglobulin G, molecular weight: 150,000) was the supernatant of the culture medium after antibody production by cells derived from Chinese hamster ovaries (CHO cells), removal of CHO cells, and clarification. The pH of the culture medium, which was 7.4, was adjusted to 4.5 using a 5 mol / L aqueous acetic acid solution. 30 mL of the prepared culture medium was added to an affinity chromatography column (trade name "MabSelect SuRetm LX", manufactured by GE Healthcare Life Sciences, containing adsorbent (I) as an affinity adsorbent) and washed with 20 mmol / L phosphate-buffered saline in the adsorbed state. Desorption was performed using 100 mmol / L acetic acid with 150 mmol / L sodium chloride dissolved in it to obtain batches of adsorbent treatment solution. The obtained adsorbent was diluted 10-fold with MilliQ water in batches, and the pH was adjusted to 3.5 with 1 mol / L hydrochloric acid. The solution was then allowed to stand at 25°C for 2 hours. Next, the pH was adjusted to 5.0 with 1 mol / L Tris solution, and the solution was filtered to obtain the virus-inactivated solution. The obtained virus-inactivated solution was added to an anion exchange chromatography column (trade name "ChromSpeed ​​Q103", manufactured by Mitsubishi Chemical Corporation) equilibrated with 20 mmol / L trihydrochloric acid buffer (pH 8.5). After addition, 5 column volumes of equilibration buffer were bubbled into the column. The non-adsorbed fraction was used as the ChromSpeed ​​Q103 eluent. The obtained ChromSpeed ​​Q103 eluent was used as a three-stage purification sample in the same way as in Example 7, obtained by operating the cation exchange chromatography column. The culture supernatant, synthetic adsorbent treatment solution, and three-stage purification solution were analyzed in the same manner as in Example 7.

[0107] The results of Example 7 and Comparative Example 2 are shown in Table 4.

[0108] [Table 4] Types of synthetic adsorbents Types of liquids HCP concentration [ng / mL] antibody purity [HCP / Mab ppm] Cumulative antibody recovery rate [%] Example 7 Synthetic adsorbent (H) Culture medium supernatant 624732 277137 100 Treatment fluid 927 523 82 Three-stage refinement 34 8 70 Comparative Example 2 Synthetic adsorbent (I) Culture medium supernatant 505852 249895 100 Treatment fluid 340 177 84 Three-stage refinement 30 9 70

[0109] As can also be seen from Table 4, compared with the affinity adsorbent (I) used in Comparative Example 2, the synthetic adsorbent (H) manufactured in Example 7, although lacking antibody adsorption / desorption and washing steps in the first stage of adsorbent treatment like adsorbent (I), can be crudely purified to an antibody concentration of three digits ppm, with a recovery rate of over 80%. In the final sample of the three-stage purification, the HCP removal rate and antibody recovery rate were equivalent to those of the step using the affinity adsorbent (I).

[0110] [Example 8] (Preparation of Culture Supernatant) The culture medium used for the monoclonal antibody (immunoglobulin G, molecular weight: 150,000) was culture supernatant 1 (antibody concentration: 2.4 mg / mL, conductivity: 20 mS / cm) obtained by producing antibodies from cells (CHO cells) derived from Chinese hamster ovaries and then removing the CHO cells. (Desalting Treatment) 0.4 g of anion exchange resin (Diaion (registered trademark) "UBA120OH", manufactured by Mitsubishi Chemical Corporation) and 0.4 g of cation exchange resin (Diaion (registered trademark) "UBK10H", manufactured by Mitsubishi Chemical Corporation) were added to 4 mL of the culture supernatant 1. Then, the pH of the culture supernatant 1, which was 7.4, was adjusted to 4.5 using a 0.1 mol / L sodium hydroxide aqueous solution. Then, the anion exchange resin and cation exchange resin were removed using a filter membrane (manufactured by Merck, 0.22 μm pore size) to obtain a desalination solution (conductivity 3 mS / cm). (Purification using synthetic adsorbent) Subsequently, 0.3 g of synthetic adsorbent (B) and 4 mL of the prepared desalination solution were measured into a container and shaken at 25°C for 8 hours. Then, the synthetic adsorbent (B) was removed by centrifugation and a filter membrane (manufactured by Merck, 0.22 μm pore size) to obtain a treatment solution based on synthetic adsorbent (B). The evaluation results of the treatment solution are shown in Table 5.

[0111] [Example 9] Except that culture supernatant 2, in which the antibody concentration of the monoclonal antibody was adjusted to 15 mg / mL, was used instead of culture supernatant 1, the same steps as in Example 8 were performed. The evaluation results of the treatment solution are shown in Table 5.

[0112] [Example 10] The treatment was performed using the same steps as in Example 8, except that desalination was not performed. The evaluation results of the treated solution are shown in Table 5.

[0113] [Example 11] The treatment was performed using the same steps as in Example 9, except that desalination was not performed. The evaluation results of the treated solution are shown in Table 5.

[0114] [Table 5] Types of synthetic adsorbents pH antibody concentration [mg / mL] electrical conductivity [mS / cm] HCP concentration [ng / mL] LRV antibody recovery rate [%] Culture medium supernatant 1 - 7.4 2.4 20 508417 - - Culture medium supernatant 2 - 7.4 15 20 394195 - - Example 8 Synthetic adsorbent (B) 4.5 2.4 3 3412 2.4 89 Example 9 4.5 15 3 995 2.9 94 Example 10 4.5 2.4 20 5352 2.2 86 Example 11 4.5 15 20 12009 1.9 88

[0115] As can also be seen from Table 5, compared with the processes implemented in Examples 10 and 11 without desalting treatment, the HCP concentration, LRV, and antibody recovery rate of Examples 8 and 9, which underwent desalting treatment, were superior. In particular, the HCP concentration, LRV, and antibody recovery rate were significantly superior when the antibody concentration in the culture medium was high.

[0116] [Example 12] Except for the desalting treatment of the culture supernatant as described in Example 8, the use of synthetic adsorbent (B) instead of synthetic adsorbent (H), and the undiluted virus inactivation solution, the same treatment as in Example 7 was performed. The evaluation results are shown in Table 6 together with the results of Example 7 and Comparative Example 2.

[0117] [Table 6] Types of synthetic adsorbents Types of liquids HCP concentration [ng / mL] antibody purity [HCP / Mab ppm] Cumulative antibody recovery rate [%] Example 7 Synthetic adsorbent (H) Culture medium supernatant 624732 277137 100 Treatment fluid 927 523 82 Three-stage refinement 34 8 70 Example 12 Synthetic adsorbent (B) Culture medium supernatant 1031731 416021 100 Desalination solution 733652 380926 93 Treatment fluid 1043 727 90 Three-stage refinement 81 twenty three 81 Comparative Example 2 Synthetic adsorbent (I) Culture medium supernatant 505852 249895 100 Treatment fluid 340 177 84 Three-stage refinement 30 9 70

[0118] As can also be seen from Table 6, compared with Example 7, in Example 12, by incorporating the desalting step (step (A)) into the process, the four-fold dilution using MilliQ water before anion exchange chromatography can be omitted, and the antibody recovery rate after three-stage purification is also a higher value of 81%. In addition, the final sample after three-stage purification shows the same HCP removal rate as the step using the affinity adsorbent, i.e., adsorbent (I).

[0119] This disclosure has been described in detail using specific methods, but it will be apparent to those skilled in the art that various modifications can be made without departing from the intent and scope of this disclosure. This application is based on Japanese Patent Application No. 2021-097149, filed on June 10, 2021, the entire contents of which are incorporated herein by reference. [Simplified Explanation of the Diagram]

[0017] Figure 1 is a graph showing the measured values ​​of the differential pore volume (mL / g) of the synthetic adsorbents (A) to (G) in the examples and comparative examples.

Claims

1. A synthetic adsorbent, wherein the maximum value of the differential pore volume (mL / g) under pressure conditions of 0.5 psia to 30.0 psia, as determined by the following method, exceeds 0.05 mL / g, <Method for determining differential pore volume (mL / g)> 1. A sample container containing the dried synthetic adsorbent is placed under reduced pressure to below 10 Pa, and mercury as specified in Japanese Industrial Standard K8572 is depressurized to below 10 Pa and degassed, and then filled into the sample container at a pressure of 0.5 psia; 2. For a sample container filled with the mercury, the pressure is gradually increased from 0.5 psia to 30.0 psia, and the amount of mercury injected into the synthetic adsorbent at this time is measured.

3. Calculate the differential pore volume (mL / g), which is obtained by dividing the increase in mercury injection when the pressure increases by one stage, calculated based on the mercury injection amount determined in step 2, by the measured synthetic adsorption dose.

2. The synthetic adsorbent as described in claim 1 is a synthetic adsorbent for antibody purification.

3. The synthetic adsorbent as claimed in claim 1 or 2, having fine pores.

4. The synthetic adsorbent as claimed in claim 3, wherein the radius of the pores is 3 nm to 15 nm.

5. The synthetic adsorbent as described in claim 1 or 2 is hydrophobic.

6. The synthetic adsorbent as claimed in claim 1 or 2, comprising at least one selected from styrene-based resins and acrylic-based resins.

7. The synthetic adsorbent as claimed in claim 1 or 2, wherein the volume average particle size is 1 μm to 500 μm.

8. The synthetic adsorbent as claimed in claim 1 or 2, wherein the corrected roundness value obtained by the following method exceeds 0.10; <Method for determining and calculating the corrected roundness value> 1. Photograph the synthetic adsorbent using an optical microscope; 2. Use the image analysis software WinROOF2018 to create an approximate circle of the photographed synthetic adsorbent; 3. According to Japanese Industrial Standard B0621, clamping is performed using two concentric geometric circles (two concentric circles) that are concentric with the approximate circle; 4. The difference between the radius of the outer circle and the radius of the inner circle in the concentric circles where the interval between the two concentric circles in step 3 is minimized is taken as the roundness.

5. Divide the calculated roundness by the radius of the approximate circle; 6. Perform the measurements and calculations described in steps 1 to 5 on more than 100 points, and use the average value as the corrected roundness.

9. The synthetic adsorbent as claimed in claim 1 or 2, wherein the crushing strength is 100 gf / particle to 2000 gf / particle.

10. The synthetic adsorbent as claimed in claim 2, wherein the antibody is a monoclonal antibody.

11. The synthetic adsorbent as claimed in claim 10, wherein the monoclonal antibody has a molecular weight of 100,000 or more.

12. The synthetic adsorbent as claimed in claim 10 or 11, wherein the monoclonal antibody is immunoglobulin G.

13. A method for purifying an antibody, wherein a mixture comprising the antibody and impurities is mixed with a synthetic adsorbent as described in claim 1 and then filtered.

14. A method for purifying an antibody, comprising: The step of passing a mixture containing antibodies and impurities through a synthetic adsorbent as described in claim 1 to recover non-adsorbed fractions containing antibodies that were not adsorbed by the synthetic adsorbent.

15. A method for purifying an antibody, comprising the following steps (i) and (ii): Step (i): a contacting step, wherein a mixed solution containing the antibody and impurities is contacted with a synthetic adsorbent as described in claim 1; Step (ii): a separation step, wherein, after step (i), the mixed solution is separated from the synthetic adsorbent.

16. The method for purifying an antibody as claimed in claim 15, wherein the preceding part of step (i) includes the following step (A): Step (A): an ion exchange resin contacting step, wherein a mixed solution containing the antibody and impurities is contacted with at least one selected from the group consisting of anion exchange resins and cation exchange resins.

17. A method for purifying an antibody as claimed in any one of claims 13 to 16, wherein the impurity comprises a substance with a molecular weight of less than 50,000.

18. A method for purifying an antibody as claimed in any one of claims 13 to 16, wherein the impurity comprises at least one selected from the group consisting of proteins derived from host cells, polymers derived from antibodies, degradation products derived from antibodies, and nucleic acids.

19. A method for purifying an antibody as claimed in any one of claims 13 to 16, wherein the mixture is mixed with a synthetic adsorbent in a liquid with a pH of 3 to 8, the mixture is passed through the synthetic adsorbent, or the mixed solution is contacted with the synthetic adsorbent.

20. A method for manufacturing an antibody, comprising a method for purifying the antibody as described in any one of claims 13 to 16.

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