Ph-responsive copolymer and antibody separation column packing material containing the same

Abstract

To provide a novel material excellent in durability and capable of being produced at low cost, which is useful for antibody separation, and to provide means for separating antibodies using the material.SOLUTION: A pH-responsive copolymer is represented by the following general formula (I) (where R1, R2, R3, and R4 independently denote organic groups constituting a main chain; R5 denotes a cationic group containing an imidazole skeleton and exhibiting pH responsiveness; R6 denotes an anionic group exhibiting pH responsiveness; R7 denotes an uncharged organic group; R8 denotes a crosslinkable group; l represents an integer of 1 or more; m represents an integer of 1 or more; n represents an integer of 0 or more; o represents an integer of 0 or more; and each of R1 to R8 in the repeating units may be of a single type or a plurality of types), and the charge changes depending on the pH.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pH-responsive copolymer and a column packing material containing the same, particularly a column packing material for antibody separation. [Background technology]

[0002] Antibodies have traditionally been widely used as reagents for antigen testing and antibody medicines. Among antibodies, monoclonal antibodies derived from a single clone are widely used because they consist only of antibodies with the same properties. Monoclonal antibodies are usually purified from hybridoma culture fluid or the ascites of animals transplanted with hybridomas. Protein A columns are widely used for this purification (Non-Patent Document 1). Protein A is a protein that has a high affinity for the Fc region of IgG antibodies and can bind to a wide range of antibodies.

[0003] Antibody antigen-binding fragments, such as Fab and scFv, have the same antigen-binding ability as antibodies and have recently attracted attention as biopharmaceuticals and diagnostic agents. Antibody fragments have smaller molecular size than antibodies, allowing for easier cell penetration, rapid extracorporeal clearance, and low formulation viscosity. Furthermore, expression systems using Escherichia coli as a host are widely used to produce antibody fragments, allowing for simple genetic manipulation and cost-effective production. However, the purification process for antibody fragments remains a significant challenge, hindering large-scale, low-cost production.

[0004] While protein A affinity chromatography is the most widely used initial recovery step for antibody purification, antibody fragments lack the Fc region that protein A binds to, making protein A incompatible. Therefore, antibody fragments are typically purified through multiple steps, combining protein L affinity chromatography with ion exchange chromatography. Protein L is a protein expressed on the surface of Peptostreptococcus magnus bacteria, and its B domain binds to Ig light chains. However, protein L only exhibits affinity for κ light chains, making it incompatible with IgGs containing λ light chains. On the other hand, the camelid affinity ligand (LamdaFabSelect) only exhibits affinity for λ light chains and can be used to purify IgGs containing λ light chains. Furthermore, immobilized metal ion affinity chromatography (IMAC) can be used to purify antibody fragments tagged with peptides, such as histidine tags. However, this method presents several challenges, including the aggregation of antibody fragments caused by peptide tags and the need for tag cleavage and removal steps. As such, there are currently no methods that can be applied to purify a wide range of Fabs, and there is a need to develop a comprehensive Fab purification method.

[0005] Meanwhile, the applicant of the present application previously invented a pH-responsive copolymer and filed a patent application (Patent Document 1). However, Patent Document 1 does not describe a pH-responsive copolymer having a cationic group with an imidazole skeleton, nor does it describe the use of a pH-responsive copolymer for antibody separation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-143183 [Non-patent literature]

[0007] [Non-Patent Document 1] Low, D.; O'Leary, R.; Pujar, NS Future of antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2007, 848 (1), 48-63. Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, Protein A columns are widely used for separating antibodies. However, because Protein A is a protein, it has problems such as lack of durability and high production costs. There is a demand for a substance that can be chemically synthesized, has excellent durability, can be produced inexpensively, and can replace Protein A.

[0009] There is also a need for a simple and versatile means for separating antigen-binding fragments of antibodies.

[0010] Therefore, an object of the present invention is to provide a novel substance that is highly durable, can be produced at low cost, and is useful for separating antibodies, and a means for separating antibodies using the same.

[0011] A further object of the present invention is to provide a simple and versatile means for separating antigen-binding fragments of antibodies. [Means for solving the problem]

[0012] As a result of extensive research, the present inventors discovered that a pH-responsive copolymer comprising a unit having a cationic group with an imidazole skeleton and a unit having a pH-responsive anionic group can be attached to and detached from an antibody depending on the pH, thereby completing the present invention.

[0013] The present inventors also discovered that the pH-responsive copolymer can be attached to and detached from the antigen-binding fragment of an antibody depending on the pH, and that there is a pH at which the Fc fragment of the antibody is detached but the antigen-binding fragment remains bound, and that this can be utilized to separate the antigen-binding fragment from the Fc fragment, thereby completing the present invention.

[0014] That is, the present invention provides the following. (1) The following general formula [I]

[0015] [ka] (In the formula, R 1 , R 2 , R 3 and R 4 are, independently of each other, organic groups constituting the main chain, R 5 is a pH-responsive cationic group containing an imidazole skeleton, R 6 is a pH-responsive anionic group, R 7 is an uncharged organic group, R 8 represents a crosslinkable group, l represents an integer of 1 or more, m represents an integer of 1 or more, n represents an integer of 0 or more, and o represents an integer of 0 or more. R 1 ~R 8 may be of a single type or of multiple types) This is a pH-responsive copolymer whose charge changes depending on the pH. (2) The cationic group is represented by the following general formula:

[0016] [ka] (In each formula, R 9 is an optional spacer group, R 10 ~R 12 each independently represents hydrogen or an organic group. The pH-responsive copolymer according to (1), which is at least one selected from the group consisting of: (3) The pH-responsive copolymer according to (2), wherein the cationic group is a histamine methacrylamide residue. (4) The anionic group is represented by the following general formula:

[0017] [ka] (In the formula, R 13 is an optional spacer group, R 14 ~R 19 each independently represents hydrogen or an organic group. The pH-responsive copolymer according to (1), which is at least one selected from the group consisting of: (5) The pH-responsive copolymer according to (4), wherein the anionic group is a group containing a carboxyl group. (6) The above R 7 is a methacrylic acid ester residue or an acrylic acid ester residue. (7) R 1 , R 2 , R 3 and R 4 and are each independently an alkyl group having 2 to 4 carbon atoms. (8) The pH-responsive copolymer according to (1), wherein the crosslinkable group is a group containing an ethylenically unsaturated bond. (9) The pH-responsive copolymer according to any one of (1) to (8), wherein a plurality of molecules are crosslinked by crosslinking between the crosslinkable groups. (10) The above R 1 If there are 100 moles of units containing R 2 20 to 500 moles of units containing R 3 The unit containing R is 0 to 2000 moles. 4 The pH-responsive copolymer according to (1), wherein the number of units comprising the following is 0 mole to 400 moles. (11) The pH-responsive copolymer according to any one of (1) to (10), wherein one end is bound to a particle. (12) A column packing material comprising the pH-responsive copolymer according to (11). (13) The column packing material according to (12), which is a column packing material for antibody separation. (14) A method for separating antibodies, comprising the steps of: applying a sample solution containing an antibody to the antibody separation column packing material described in (12) packed in a column; then applying a binding buffer to the column to remove impurities in the sample solution; and then applying an elution buffer to the column to elute the antibody in the sample solution. (15) The column packing material according to (12), which is a column packing material for separating antigen-binding fragments of antibodies. (16) A method for separating antigen-binding fragments of antibodies, comprising the steps of: applying a sample solution containing antigen-binding fragments of antibodies to a column packing material for separating antigen-binding fragments of antibodies according to claim 15 packed in a column; then applying a binding buffer to the column to remove impurities in the sample solution; and then applying an elution buffer to the column to elute the antigen-binding fragments of antibodies in the sample solution, wherein the pH of the binding buffer is such that a majority of the antigen-binding fragments of interest remain bound to the column packing material. (17) The method according to (16), wherein the pH of the binding buffer is 6.0 to 7.5. (18) The method according to (17), wherein the antigen-binding fragment is a Fab fragment. [Effects of the Invention]

[0018] The present invention provides a novel copolymer with pH-responsiveness. The pH-responsive copolymer of the present invention binds to antibodies in the neutral range and dissociates from them in the acidic range, making it useful for antibody separation. The pH-responsive copolymer of the present invention can be easily chemically synthesized by addition polymerization of monomers having unsaturated double bonds, resulting in low production costs. Furthermore, as a synthetic copolymer, it is more durable than proteins. Furthermore, the pH-responsive copolymer of the present invention makes it possible to separate the antigen-binding fragment of an antibody from the Fc fragment. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 2 is a diagram for schematically explaining a method for preparing resin particles carried out in the following examples. [Figure 2] FIG. 1 shows the results of absorbance measurements obtained in the following examples. [Figure 3] 3A and 3B show the results of the study of the amount of polymer modification carried out in the following examples. In Fig. 3A, the peak and the area with higher intensity after 2-3 minutes show the results for 10 mM PB at pH 7. In Fig. 3B, the area with a peak shows the results for 10 mM PB at pH 7. [Figure 4] 1 shows the elution profile of benzene obtained in the following Examples, with the results for tBMA-2, tBMA-3, and tBMA-1 shown in descending order of peak height. [Figure 5] These figures show the results of basic property evaluations obtained in the following examples. In Figures 5(b), 5(c), and 5(f), the results for EEMA, EMA, and tBMA are shown in descending order of peak height. In Figure 5(d), the high peaks for EEMA and EMA almost overlap, and the low peaks represent the results for tBMA. In Figure 5(e), the results for EEMA, tBMA, and EMA are shown in descending order of peak height. [Figure 6] 6(a), (b), and (c) show the relationship between the salt concentration of the eluent and the elution behavior obtained in the following examples. The results for 50 mM, 100 mM, and 10 mM are shown in descending order of peak height. [Figure 7] 7(a) and 7(c) show the results of gradient elution of Rituximab obtained in the following examples. In Fig. 7(a) and Fig. 7(c), the results for EEMA, EMA, and tBMA are shown in descending order of peak height. In Fig. 7(b), the results for EEMA, tBMA, and EMA are shown in descending order of peak height. [Figure 8] FIG. 1 shows the results of gradient elution from a HisMA20-tBMA column obtained in the following example. [Figure 9] FIG. 1 shows the results of recovery rate of Rituximab obtained in the following Example. [Figure 10] FIG. 1 shows the results of a retention capacity test obtained in the following example. [Figure 11] FIG. 1 shows the results of gradient elution of mouse IgG obtained in the following example. [Figure 12] FIG. 1 shows the results of purification of Rituximab from a mixture with BSA, performed in the following Examples. [Figure 13] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with BSA, performed in the following Examples. [Figure 14] FIG. 1 shows the results of purification of Rituximab from a mixture with mouse ascites fluid, performed in the following Examples. [Figure 15] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with mouse ascites fluid, obtained in the following Examples. [Figure 16] FIG. 1 shows the results of purification of Rituximab from a mixture with hybridoma cell culture supernatant, performed in the following Examples. [Figure 17] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with hybridoma cell culture supernatant, obtained in the following Examples. [Figure 18] FIG. 1 shows the recovery rates of Rituximab obtained in the following Examples. [Figure 19] FIG. 1 shows the results of purification of Trastuzumab from a mixture with BSA, performed in the following Examples. [Figure 20] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with BSA, obtained in the following Examples. [Figure 21] FIG. 1 shows the results of purification of Trastuzumab from a mixture with mouse ascites fluid, performed in the Examples below. [Figure 22] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with mouse ascites fluid, obtained in the following Examples. [Figure 23] FIG. 1 shows the results of purification of Trastuzumab from a mixture with hybridoma cell culture supernatant, performed in the Examples below. [Figure 24]FIG. 1 shows the results of SEC analysis of fractions collected from a mixture with hybridoma cell culture supernatant, obtained in the following Examples. [Figure 25] FIG. 1 shows the recovery rates of Trastuzumab obtained in the following Examples. [Figure 26] 26(a) and (b) show the results of purifying Rituximab from a mixture with hybridoma cell culture supernatant, performed in the following Examples. In Fig. 26(a) and (b), the results are shown for Rituximab concentrations of 0.2 mg / mL, 2 mg / mL, and 0.02 mg / mL, in descending order of peak height. [Figure 27] FIG. 1 shows the recovery rates of Rituximab obtained in the following Examples. [Figure 28] FIG. 1 shows the results of SEC analysis of fractions collected from a mixture of hybridoma cell culture supernatant (0.2, 0.02 mg / mL Rituximab) obtained in the following Examples. [Figure 29] 29 shows the results of gradient elution of Rituximab performed in the following example. In Fig. 29, the peaks show the results for washing times of 3 minutes, 10 minutes, and 20 minutes, from the left. [Figure 30] FIG. 1 shows the results of column durability and reusability tests carried out in the following examples. [Figure 31] FIG. 1 shows the results of a maximum antibody binding capacity test obtained in the following Examples. [Figure 32] FIG. 1 shows comparative results of SEC analysis before and after digestion with papain, obtained in the following Examples. [Figure 33] FIG. 1 shows the results of purity evaluation of isolated antibody fragments obtained in the following examples. [Figure 34] FIG. 1 shows the effect of mobile phase pH on the retention of antibody fragments, obtained in the following Examples. [Figure 35] FIG. 1 shows the results of chromatography when Fab fragments were purified from digested samples of each antibody drug, obtained in the Examples below. DETAILED DESCRIPTION OF THE INVENTION

[0020] As described above, the pH-responsive copolymer of the present invention has a structure represented by the above general formula [I].

[0021] In the above general formula [I], R 1 , R 2 , R 3 and R 4 are each independently an organic group constituting the main chain, and are preferably represented by the general formula [IV]

[0022] [ka]

[0023] In the general formula [IV], R 20 , R 21 and R 22 are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms (including branched alkyl groups when the number of carbon atoms is 3 or more, the same applies hereinafter), and are preferably hydrogen or a methyl group. As will be described later, the copolymer of the present invention can be easily produced by addition polymerization of (meth)acrylic monomers such as (meth)acrylic acid and (meth)acrylamide, vinyl ester monomers, or vinyl amide monomers (hereinafter sometimes referred to as "vinyl monomers") having a cationic group, an anionic group, or an organic group, as will be described later. In these cases, R 10 is hydrogen or a methyl group, R 11 and R 12 becomes hydrogen. Vinyl monomers such as (meth)acrylic acid and (meth)acrylamide are advantageous in that any group can be easily bonded to the carboxyl group or amide group they contain. 1 , R 2 , R 3 and R 4 , may be the same or different, and R 1 , R 2 , R 3and R 4 If there are multiple instances of each of R 1 , R 2 , R 3 and R 4 may be the same or different.

[0024] In the above general formula [I], R 5 represents a cationic group containing an imidazole skeleton that is pH-responsive. The pH-responsive cationic group is preferably a group that does not have a charge in the neutral range, but has a positive charge due to the addition of a hydrogen atom in the vicinity of weak acidity. Preferred examples of such cationic groups include:

[0025] [ka]

[0026] In these general formulas, R 9 is an optional spacer group, R 10 ~R 12 R each independently represents hydrogen or an organic group. 9 If there is R 9 R may be any structure that connects the main chain and the imidazole skeleton. 9 When a copolymer is synthesized by polymerization of a vinyl monomer, the copolymer will have one of the structures of the vinyl monomer residues described above at the end closer to the main chain, but if no vinyl monomer is used in the synthesis, these residues may not be present. 9 Among them, the structure of the portion other than the vinyl monomer residue is not particularly limited, but preferred examples include alkylene groups having 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms. These alkylene groups may be substituted with a substituent such as an alkyl group having 1 to 6 carbon atoms, and may also contain at least a portion of an alkylene glycol structure in which a carbon atom is replaced with an oxygen atom, an imino structure in which a carbon atom is replaced with a nitrogen atom, or a carbonyl group in which ═O is bonded to a carbon atom. Furthermore, R 9may contain a cycloalkylene group having 3 to 10 carbon atoms, preferably 4 to 8 carbon atoms, and the carboxyl group may be bonded to this cycloalkylene group. The cycloalkylene group may be substituted with 1 to 4 substituents such as alkyl groups having 1 to 6 carbon atoms. In each of the above general formulas showing the preferred pH-responsive cationic groups, R 10 ~R 12 Preferred examples of the cationic group include hydrogen and alkyl groups having 1 to 6 carbon atoms, and more preferred examples include hydrogen and alkyl groups having 1 to 3 carbon atoms. Particularly preferred examples of the cationic group include the histamine methacrylamide residue (R 10 ~R 12 are all hydrogen, R 9 Examples include -C(O)-NH-CH2-CH2-.

[0027] In the above general formula [I], R 6 represents a pH-responsive anionic group. The pH-responsive anionic group is preferably a group that does not have a charge in the vicinity of weak acidity, but releases a hydrogen atom to have a negative charge in the neutral region. Preferred examples include groups represented by the following general formula:

[0028] [ka]

[0029] In each of these general formulas, R 13 is an optional spacer group, R 14 ~R 19 R each independently represents hydrogen or an organic group. 13 When a spacer group represented by 13 For an explanation of R 9 The explanation for the spacer group represented by R 13 ~R 18Preferred examples of the anionic group include hydrogen and alkyl groups having 1 to 6 carbon atoms, and more preferably hydrogen and alkyl groups having 1 to 3 carbon atoms. Particularly preferred anionic groups are groups containing a carboxyl group (groups represented by the leftmost general formula of the above five general formulas), and in particular, a carboxyl group (without a spacer group) is preferred.

[0030] In the above general formula [I], R 7 is any organic group, preferably an uncharged organic group. Here, the uncharged organic group may be any group that has no charge within the pH range of 2 to 12, at least within the pH range of 3 to 7. The uncharged organic group does not have to be present (i.e., n in the general formula [I] is 0), but its presence increases the hydrophobicity of the copolymer as a whole and strengthens the interaction with the antibody, so its presence is preferred. 7 When a copolymer is synthesized by polymerization of a vinyl monomer, the copolymer will have one of the structures of the vinyl monomer residues described above at the end closest to the main chain, but if no vinyl monomer is used in the synthesis, these residues may not be present. 7 A preferred example of is a (meth)acrylic acid ester residue, and in this case, the group bonded to oxygen in -C(O)-O- constituting the ester can be an alkyl group having 1 to 6 carbon atoms. A particularly preferred example is an ethoxyethyl group used in the examples below.

[0031] In the above general formula [I], R 8 represents a crosslinkable group. Examples of the crosslinkable group include groups containing an ethylenically unsaturated bond, and a preferred example is a (meth)acrylic acid residue that maintains the ethylenically unsaturated bond. For example, the crosslinkable group may include a group that contains a (meth)acrylic acid residue in the form of an ester bond to a (meth)acrylic acid residue bonded to the main chain, as used in the examples below. 8 may not be present (when o is 0 in the above general formula [I]).

[0032] R 8When R is present in a single copolymer molecule, the crosslinking between the crosslinkable groups of the multiple copolymer molecules results in a crosslinked configuration of multiple copolymer molecules. 4 When multiple R are present, they are 8 They may also be linked together to form a ring moiety.

[0033] The copolymer of the present invention may be a random polymer or a block polymer. Random polymers are preferred because they can be produced simply by polymerizing each monomer, as specifically described in the following examples.

[0034] In the copolymer of the present invention, R 1 If there are 100 moles of units containing R 2 The unit containing R is 20 to 500 moles, more preferably 80 to 200 moles. 3 The unit containing R is 0 to 2000 moles, more preferably 200 to 800 moles. 4 The amount of the unit containing the following is preferably 0 to 400 moles, more preferably 3 to 30 moles.

[0035] The mass average molecular weight of the copolymer of the present invention (uncrosslinked or before crosslinking) is not particularly limited, but is usually about 1,000 to 1,000,000, and preferably about 5,000 to 100,000.

[0036] The copolymer of the present invention may be in a form in which one end is bound to a particle. A particle in which one end of the copolymer of the present invention is bound can be preferably used as a column packing material, particularly as a column packing material for antibody separation. Examples of particles include resin particles, silica particles, and magnetic nanoparticles. Among these, various commercially available resin particles used as column packing materials are particularly preferred. Examples of preferred resin particles include the TOYOPERARL series (trade name, manufactured by Tosoh Corporation), which are hydrophilic vinyl polymers. Some of these commercially available resin particles have functional groups such as amino groups or carboxyl groups bound to their surfaces. The copolymer molecules of the present invention can be easily bound to such functional group-bound resin particles. The copolymer can be bonded to the resin particles after synthesis, or can be bonded to the resin particles simultaneously with the copolymer synthesis (addition polymerization reaction) (see Examples below). The copolymer can be bonded to a membrane or monolith instead of the particles, or the copolymer itself can be used as a column packing material without being bonded to particles, membranes, or monoliths.

[0037] The copolymers of the present invention, preferably those in a form in which one end is bound to a particle or membrane, can be used as column packing materials, particularly as column packing materials for antibody separation. A method for separating antibodies using such a column packing material for antibody separation includes the steps of applying an antibody-containing sample solution to the column packed with the antibody separation column packing material, then applying a binding buffer to the column to remove impurities from the sample solution, and then applying an elution buffer to the column to elute the antibody in the sample solution. The pH of the binding buffer is typically about 6 to 10, preferably about 7 to 9, and the pH of the elution buffer is typically about 3 to 7, preferably about 3 to 5. A preferred example of the antibody separation method is described in detail in the Examples below.

[0038] The column packing materials described above can also be used as column packing materials for separating antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include Fab fragments, (Fab')2 fragments, scFv fragments, diabody fragments, and domain antibody (dAb) fragments. Of these, Fab fragments are preferred. As specifically demonstrated in the Examples below, there exists a pH at which the majority of antigen-binding fragments remain bound to the column packing material. By washing the column with a buffer having such a pH, the antigen-binding fragments remain bound to the column packing material, while impurities that do not bind to the column packing material are eluted from the column at this pH. Here, "the majority" means 60% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. The pH at which the majority of antigen-binding fragments remain bound to the column packing material varies depending on the type of antibody and the type of antigen-binding fragment. Therefore, the appropriate pH of the binding buffer can be determined by conducting preliminary experiments such as those specifically described in the Examples below. For example, as specifically described in the Examples below, when the antigen-binding fragment is a Fab fragment and contains an Fc fragment generated by papain digestion of an antibody as a contaminant, a sample containing the Fab and Fc fragments is applied to a column, followed by application of a binding buffer having a pH of approximately 6.0 to 7.5, preferably approximately 6.3 to 6.7, and most preferably 6.5, to the column, thereby eluting the Fc fragment while leaving most of the Fab fragment bound to the column packing. The antigen-binding fragment (Fab fragment in the Examples below) can then be eluted and recovered from the column by applying an elution buffer with a pH lower than 6.0, preferably approximately 3 to 5.5. This method for separating antigen-binding fragments can be applied not only to separating the antigen-binding fragment of interest from antibody digestion products, but also to purifying antigen-binding fragments produced by genetic engineering.

[0039] The copolymer of the present invention can be easily produced by addition polymerizing addition-polymerizable monomers composed of the above-mentioned units. 5 , R6 , R 7 , R 8 (See the Examples below.) When producing a copolymer in which one end is bonded to a particle such as a resin particle having a functional group on its surface, the functional group on the particle surface is first reacted with a polymerization initiator, for example, 4,4'-azobis(4-cyanovaleric acid) (V-501 (trade name)), and then each addition-polymerizable monomer can be addition-polymerized (See the Examples below.)

[0040] The present invention will be described in detail below based on examples. However, the present invention is not limited to the following examples. In the following examples, the TOYOPEARL used was TOYOPEARL AF-Amino-650M, which is the trade name of a commercially available product manufactured by Tosoh Corporation.

[0041] Example 1 Preparation of copolymer 4.2.1 Synthesis of HisMA 4.2.1.1 Synthesis of Boc-HisMA

[0042] [ka]

[0043] Under an argon atmosphere, histamine dihydrochloride (4.00 g, 21.54 mmol) and ultrapure water (20 mL) were added to a 200 mL three-necked eggplant. The mixture was then cooled to 0 °C, and a solution of methacryloyl chloride (3.50 mL, 34.46 mmol) in anhydrous CHCl (20 mL) and 3.88 M aqueous NaOH (20 mL) were added dropwise over 30 min. The mixture was stirred at room temperature for 21 h. After completion of the reaction, the solvent was removed by evaporation under reduced pressure and lyophilization to yield a mixture of the product and salt as a white solid. The resulting white solid was suspended in 50 mL of isopropanol, filtered, and washed with 50 mL of isopropanol. The filtrate was evaporated under reduced pressure to yield the crude product (4.98 g).

[0044] Under an argon atmosphere, the crude product (4.98 g) and anhydrous DMF (43 mL) were placed in a 200 mL two-necked eggplant and stirred to dissolve the crude product. The mixture was cooled to 0 °C, and di-tert-butyl dicarbonate (5.3 mL, 23.69 mmol) and triethylamine (3.6 mL, 25.85 mmol) were added. The mixture was then stirred at room temperature for 20 hours. After the reaction was complete, 120 mL of purified water was added, and the mixture was extracted three times with hexane / ethyl acetate (1 / 1), washed with saturated brine, and dried over anhydrous sodium sulfate. The filtrate was evaporated under reduced pressure to give the crude Boc-HisMA (2.80 g). This was then purified by silica gel column chromatography (hexane / ethyl acetate (1 / 3)) to give Boc-HisMA (580 mg, 2.08 mmol, 10%) as a yellow oil.

[0045] 1 H NMR (400 MHz, CD--3OD) δ(ppm) 8.13(s, 1H), 7.30(s, 1H), 5.66(s, 1H), 5.35(s, 1H), 3.49(t, J = 6.98 Hz, 2H), 2.77(t, J = 6.98 Hz, 2H), 1.92(s, 3H), 1.62(s, 9H).

[0046] 4.2.1.2 Deprotection of Boc-HisMA

[0047] [ka]

[0048] Boc-HisMA (233.11 mg, 0.835 mmol) and MeOH (8.35 mL) were added to a 100 mL eggplant and stirred to dissolve. The mixture was then cooled to 0 °C and acetyl chloride (0.6 mL, 8.35 mmol) was added, followed by stirring at room temperature for 17 hours. After completion of the reaction, the solvent was removed under reduced pressure to obtain HisMA·HCl (203.6 mg, 0.944 mmol, quantitative yield).

[0049] 1 H NMR (500 MHz, CD--3OD) δ(ppm) 8.81(s, 1H), 7.35(s, 1H), 5.67(s, 1H), 5.37(s, 1H), 3.55(t, J = 6.73 Hz, 2H), 2.95(t, J = 6.73 Hz, 2H), 1.91(s, 3H).

[0050] 4.2.2 Introduction of initiator into TOYOPEARL AF-Amino-650M

[0051] [ka]

[0052] Approximately 10 mL of DMF was added to a 100 mL two-necked flask. The polymerization initiator, 4,4'-Azobis(4-cyanovaleric acid) (V-501, 1169 mg, 3.75 mmol), and the condensation agent, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 2063 mg, 7.5 mmol), were then dissolved. Finally, TOYOPEARL AF-Amino-650 M (particle size: 65 μm, pore size: 100 nm, 2.00 g) was added, and the DMF solvent was added until the total volume reached 30 mL. After 30 minutes of nitrogen gas bubbling, the mixture was allowed to react at room temperature for 22 hours while shaking on a shaker. After the reaction, the reaction mixture was filtered by suction, washed with ethanol (150 mL), substituted with acetone, and then vacuum dried in a desiccator to obtain V-501-bound TOYOPEARL (1.94 g).

[0053] 4.2.3 Confirmation of V-501 incorporation by ninhydrin reaction The binding of V-501 to TOYOPEARL was confirmed by the ninhydrin reaction.

[0054] 4.2.3.1 Solution preparation (a) Ninhydrin solution Ninhydrin (356.3 mg, 2.0 mmol) was added to 20 mL of ultrapure water and dissolved under heating to prepare a 0.1 M ninhydrin aqueous solution.

[0055] (b) Benzyl triethylammonium chloride (BTEAC) solution BTEAC (455.6 mg, 2.00 mmol) was dissolved in 10 mL of ultrapure water to prepare a 0.2 M BTEAC solution.

[0056] 4.2.3.2 Ninhydrin reaction Two 2 mg portions of TOYOPEARL before and after V-501 loading were weighed into two separate screw cap tubes, and 400 μL of ethanol was added. The particles were left to swell for 10 minutes. 100 μL of 0.1 M ninhydrin solution was added and mixed for 5 minutes with ultrasound. The mixture was heated in a 95°C oil bath for 10 minutes. 100 μL of 0.2 M BTEAC solution was then added to confirm that the dye had not been adsorbed to the resin. 500 μL of ethanol was added, and the solution in the screw cap tube was transferred to a centrifuge tube and centrifuged at 8000 rpm for 3 minutes at room temperature. The supernatant was collected, and 1 mL of ethanol was added. The mixture was centrifuged in the same manner. The supernatant was collected in a 2 mL volumetric flask, and a small amount of ethanol was added to make up to 2 mL. The absorbance (450-750 nm) was measured.

[0057] 4.2.4 Introduction of pH-responsive mixed-charge moieties 4.2.4.1 HisMA20-tBMA To examine the difference in antibody retention depending on the amount of modification, beads with a ratio of TOYOPEARL weight to the total weight of the modifying monomer of 4:1 and beads with a ratio of 1:1 were prepared.

[0058] 4.2.4.1.1 HisMA20-tBMA-1 (TOYOPEARL / monomer = 4 / 1, w / w)

[0059] [ka] (where a, b, c, and d are the number of each unit, [ ] n indicates that it is crosslinked with other copolymer molecules. (The same applies below.)

[0060] A 100 mL two-necked flask was charged with approximately 10 mL of HPLC-grade methanol and dissolved histamine methacrylamide hydrochloride (HisMA·HCl, 15.0 mg, 0.070 mmol), MAA (6.05 mg, 0.070 mmol), tBMA (28.68 mg, 0.202 mmol), and ethylene glycol dimethacrylate (EGDMA, 1.33 mg, 0.007 mmol). Finally, 204.15 mg of V-501-conjugated TOYOPEARL was added, and the solvent was added until the total volume reached 30 mL. After 30 minutes of nitrogen gas bubbling, the mixture was incubated in an oil bath (70 °C) for 5 hours while shaking. After the reaction, the mixture was filtered, washed with methanol, purged with acetone, and vacuum-dried in a desiccator to obtain HisMA20-tBMA particles (190.5 mg).

[0061] 4.2.4.1.2 HisMA20-tBMA-2 (TOYOPEARL / monomer = 1 / 1, w / w)

[0062] [ka]

[0063] A 100 mL two-neck flask was charged with approximately 10 mL of HPLC-grade methanol and dissolved histamine methacrylamide hydrochloride (HisMA·HCl, 77.73 mg, 0.360 mmol), MAA (31.00 mg, 0.360 mmol), tBMA (148.42 mg, 1.044 mmol), and ethylene glycol dimethacrylate (EGDMA, 7.15 mg, 0.036 mmol). Finally, 264.63 mg of V-501-conjugated TOYOPEARL was added, and the solvent was added until the total volume reached 30 mL. After 30 minutes of nitrogen gas bubbling, the mixture was incubated in an oil bath (70 °C) for 5 hours. After the reaction, the mixture was filtered, washed with methanol, purged with acetone, and vacuum-dried in a desiccator to obtain HisMA20-tBMA particles (269.25 mg).

[0064] 4.2.4.2 HisMA20-EMA

[0065] [ka]

[0066] A 100 mL two-neck flask was charged with approximately 10 mL of HPLC-grade methanol and dissolved histamine methacrylamide hydrochloride (HisMA·HCl, 42.32 mg, 0.196 mmol), MAA (16.89 mg, 0.196 mmol), EMA (64.84 mg, 0.569 mmol), and ethylene glycol dimethacrylate (EGDMA, 3.80 mg, 0.020 mmol). Finally, 128.24 mg of V-501-conjugated TOYOPEARL was added, and the solvent was added until the total volume reached 30 mL. After 30 minutes of nitrogen gas bubbling, the mixture was incubated in an oil bath (70 °C) for 5 hours while shaking. After the reaction, the mixture was filtered, washed with methanol, purged with acetone, and vacuum-dried in a desiccator to obtain HisMA20-EMA particles (117.04 mg).

[0067] 4.2.4.3 HisMA20-EEMA

[0068] [ka]

[0069] A 100 mL two-necked flask was charged with approximately 10 mL of HPLC-grade methanol and dissolved in histamine methacrylamide hydrochloride (HisMA·HCl, 52.02 mg, 0.241 mmol), MAA (20.75 mg, 0.241 mmol), EEMA (110.66 mg, 0.700 mmol), and ethylene glycol dimethacrylate (EGDMA, 4.73 mg, 0.024 mmol). Finally, 189.04 mg of V-501-conjugated TOYOPEARL was added, and the solvent was added until the total volume reached 30 mL. After 30 minutes of nitrogen gas bubbling, the mixture was incubated in an oil bath (70 °C) for 5 hours. After the reaction, the mixture was filtered, washed with methanol, purged with acetone, and vacuum-dried in a desiccator to obtain HisMA20-EEMA particles (155.26 mg).

[0070] 4.3 HPLC measurements 4.3.1 Preparation of pH-responsive mixed-charge column (see Figure 1) The pH-responsive mixed-charge polymer-modified beads synthesized in 4.2 were packed into an empty column (φ4.6 mm×10 mm).

[0071] First, approximately 150 mg of pH-responsive mixed-charge polymer-modified beads were placed in 15 mL of slurry solvent and thoroughly dispersed by ultrasonication to form a slurry. The resulting slurry was injected into an analytical column packer and pumped at a flow rate of 10 mL / min for 1 hour using the LC-20AR pump. The pressure was adjusted so that it did not exceed 0.3 MPa. Water / methanol (90 / 10, v / v) was then pumped for 30 minutes to replace the solution in the column. After packing, any beads that did not fit into the column were collected, vacuum-dried in a desiccator, and stored refrigerated.

[0072] The slurry solvents used for packing each packing material and the liquid delivery times are shown in Table 1. However, HisMA20-tBMA-2 (TOYOPEARL / monomer = 1 / 1, w / w) was packed using three different slurry solvents in order to examine the slurry solvent.

[0073] [Table 1]

[0074] 4.3.2 Preparation of buffers and measurement samples 4.3.2.1 Antibody Samples 4.3.2.1.1 Rituximab Samples A 4 mg / mL Rituximab solution was prepared by mixing 400 μL of rituximab stock solution (10 mg / mL) with 600 μL of 10 mM PB, pH 7. The prepared rituximab solution was passed through a protein low-binding filter (Millex-GV hydrophilic PVDF) and placed in a small-volume LC vial.

[0075] 4.3.2.1.2 Trastuzumab Sample A 4 mg / mL Rituximab solution was prepared by mixing 200 μL of trastuzumab stock solution (21 mg / mL) with 800 μL of 10 mM PB, pH 7. The prepared Rituximab solution was passed through a protein low-binding filter (Millex-GV hydrophilic PVDF) and placed in a small-volume LC vial.

[0076] 4.3.2.2 Citrate buffer Citric acid (9.61 g, 0.05 mol) and trisodium citrate dihydrate (14.7 g, 0.05 mol) were dissolved in a small amount of ultrapure water and placed in a 500 mL volumetric flask. The solution was then diluted with ultrapure water to obtain a 100 mM solution. The pH was adjusted to the desired value using a pH meter to obtain citrate buffer (CAB).

[0077] 4.3.2.3 Phosphate buffer Sodium dihydrogen phosphate dihydrate (7.80 g, 0.05 mol) and disodium hydrogen phosphate (7.10 g, 0.05 mol) were each dissolved in a small amount of ultrapure water and placed in a 250 mL volumetric flask. The solution was then diluted with ultrapure water to obtain a 200 mM solution. The desired pH was then obtained by adjusting the solution with a pH meter.

[0078] 4.3.2.4 Tris-HCl buffer Tris(hydroxymethyl)aminomethane (6.06 g, 0.05 mol) was dissolved in a small amount of ultrapure water and placed in a 50 mL volumetric flask. The solution was then diluted with ultrapure water to obtain a 1 M solution. 1 M HCl was added dropwise to the 1 M Tris solution, and the pH was adjusted to 9.0 using a pH meter to obtain a pH 9 1 M Tris-HCl buffer.

[0079] 4.3.2.5 pH 6.7 0.2 M PB (0.05% NaN3) Sodium dihydrogen phosphate dihydrate (7.80 g, 0.05 mol) and disodium hydrogen phosphate (7.10 g, 0.05 mol) were each dissolved in ultrapure water and placed in a 250 mL volumetric flask. The solution was then diluted with ultrapure water to obtain a 200 mM solution. These were mixed while checking the pH with a pH meter to prepare a 0.2 M phosphate buffer solution at pH 6.7. Sodium azide was added to the prepared 0.2 M PB solution at pH 6.7 to obtain a 0.2 M PB solution at pH 6.7 (0.05% NaN3).

[0080] 4.3.3 Examination of the amount of polymer modification The retention and elution behavior of Rituximab was compared using the HisMA20-tBMA-1 and HisMA20-tBMA-2 columns prepared in 4.3.1. The mobile phases used were 10 mM PB at pH 7 and 100 mM CAB at pH 3, as prepared in 4.3.2. Each measurement was performed at a flow rate of 0.2 mL / min, with the column oven temperature at 25 °C, a sample injection volume of 10 μL, and a detection wavelength of 280 nm.

[0081] 4.3.4 Consideration of slurry solvent To investigate the slurry solvent, benzene was eluted using the three HisMA20-tBMA-2 columns prepared in 4.3.1. 40.0 mg of benzene was dissolved in 10 mL of methanol to prepare a 4 mg / mL sample. Methanol was used as the mobile phase. Each measurement was performed at a flow rate of 0.4 mL / min, with the column oven at 25 °C, a sample injection volume of 10 μL, and a detection wavelength of 255 nm.

[0082] 4.3.5 Consideration of hydrophobic monomers Hydrophobic monomers were investigated using the columns prepared in 4.3.1. For the measurements, HisMA20-tBMA, HisMA20-EMA, and HisMA20-EEMA columns packed with methanol / chloroform = 1 / 3 (v / v) as the slurry solvent were used.

[0083] 4.3.5.1 Basic characteristics evaluation To evaluate the basic characteristics, elution experiments of Rituximab were performed. The mobile phases used were CAB (pH 3 / 4 / 5, 10 / 50 / 100 mM) and PB (pH 7, 10 / 50 / 100 mM) prepared in 4.3.2. Each measurement was performed at a flow rate of 0.2 mL / min, a column oven temperature of 25 °C, a sample injection volume of 2 μL, and a detection wavelength of 280 nm.

[0084] 4.3.5.2 Gradient elution Gradient elution of Rituximab was performed. To determine the recovery rate of eluted Rituximab from the peak area, the antibody sample was first run without the column attached, and the antibody recovery rate was calculated from the peak area. The mobile phase used was 50 mM CAB at pH 3 / 4 / 5.

[0085] Next, gradient elution of Rituximab was performed using the column prepared in 4.3.1. First, without sample injection, binding buffer (pH 7, 10 mM PB) was run for 3 minutes, elution buffer (pH 3 / 4 / 5, 50 mM CAB) for 6 minutes, and stabilization buffer (pH 7, 10 mM PB) for 3 minutes. The measurement results at these times were used as background for correction. Each measurement was then performed. The measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25 °C, sample injection volume of 2 μL, and wavelength of 280 nm.

[0086] 4.3.6 Retention Capacity Test Antibody retention capacity testing was performed using a HisMA20-EEMA column. 10 μL of antibody sample (2.5, 5.0, 10 mg / mL Rituximab) was injected, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature at 25°C, and wavelength at 280 nm.

[0087] 4.3.7 Gradient elution of mouse IgG Gradient elution of mouse IgG was performed using a HisMA20-EEMA column. 10 μL of 1 mg / mL mouse IgG was injected, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). The measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm.

[0088] 4.3.8 Purification of antibody drugs from mixed samples 4.3.8.1 Preparation of mixed samples 4.3.8.1.1 Mixture of bovine serum albumin (BSA) and antibody drug First, 40.0 mg of BSA was dissolved in 10 mL of 10 mM PB, pH 7, to prepare a 4 mg / mL BSA solution. This 4 mg / mL BSA solution was mixed with equal volumes of the 4 mg / mL monoclonal antibody (Rituximab or Trastuzumab) solution prepared in 4.3.2.1 to prepare mixed samples of BSA and antibody drugs (4 mg / mL BSA + 4 mg / mL Rituximab, 4 mg / mL BSA + 4 mg / mL Trastuzumab).

[0089] 4.3.8.1.2 Mixed sample of mouse ascites and antibody drug First, the stock solution of mouse ascites was diluted 5-fold with 10 mM PB, pH 7. This mouse ascites solution was mixed with equal volumes of the 4 mg / mL monoclonal antibody (Rituximab or Trastuzumab) solution prepared in 4.3.2.1 to prepare mixed samples of mouse ascites and Rituximab (mouse ascites + 4 mg / mL Rituximab, mouse ascites + 4 mg / mL Trastuzumab).

[0090] 4.3.8.1.3 Mixed sample of hybridoma cell culture supernatant and antibody drug Hybridoma cells 1 The hybridoma cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C for 48 hours, and the culture supernatant was collected. The collected culture supernatant was mixed with equal volumes of the 4 mg / mL monoclonal antibody (Rituximab or Trastuzumab) solution prepared in 4.3.2.1 to prepare mixed samples of hybridoma cell culture supernatant and antibody drug (hybridoma cell culture supernatant + 4 mg / mL Rituximab, hybridoma cell culture supernatant + 4 mg / mL Trastuzumab).

[0091] 4.3.8.2 Purification of Antibody Drugs from Mixtures with Bovine Serum Albumin (BSA) 10 μL of the mixed sample prepared in 4.3.8.1.1 was injected onto the HisMA20-EEMA column, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3 / 5, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0092] 4.3.8.3 Purification of antibody drugs from mixtures with mouse ascites 10 μL of the mixed sample prepared in 4.3.8.1.2 was injected onto the HisMA20-EEMA column, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3 / 5, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0093] 4.3.8.4 Purification of antibody drugs from a mixture with hybridoma cell culture supernatant 10 μL of the mixed sample prepared in 4.3.8.1.3 was injected onto the HisMA20-EEMA column, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3 / 5, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0094] 4.3.8.5 Characterization of Purified Antibodies Eluted from the HisMA20-EEMA Column In a purification experiment for antibody drugs from a mixed sample, each elution peak was fractionated. The sample eluting between 0.5 and 1.5 minutes was collected as Fraction 1. In addition, the sample eluting between 6.8 and 7.8 minutes using a gradient elution with pH 3 50 mM CAB, and the sample eluting between 7.0 and 8.0 minutes using a gradient elution with pH 5 50 mM CAB, were collected as Fraction 2. However, for the fractions eluted with pH 3 50 mM CAB, 1 M Tris-HCl buffer (pH 9) was added to the collection tube in a volume equivalent to 1 / 10 of the elution volume to neutralize the eluent. The collected Fractions 1 and 2, the mixed sample before separation, and standard samples (rituximab, trastuzumab, BSA, mouse ascites, and hybridoma cell culture supernatant) were then analyzed using a size-exclusion column (SEC column). The measurements were performed using a Tosoh Bioscience TSK gel UP-SW3000 size exclusion column (4.6 mm id × 150 mm, 2 μm) with a pH 6.7 mobile phase of 0.2 M PB (0.05% NaN3) at a flow rate of 0.35 mL / min, a column oven temperature of 25 °C, a sample injection volume of 20 μL, and a wavelength of 280 nm.

[0095] 4.3.10 Purification of Rituximab from Low-Concentration Samples 4.3.10.1 Preparation of mixed samples The 4 mg / mL Rituximab solution prepared in 4.3.2.1 was diluted with 10 mM PB, pH 7, to prepare 0.4 and 0.04 mg / mL Rituximab solutions. Hybridoma cells 1The hybridoma cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C for 48 hours, and the culture supernatant was collected. The collected culture supernatant was mixed with equal volumes of 0.4 and 0.04 mg / mL Rituximab solutions to prepare hybridoma cell culture supernatant and Rituximab mixtures (hybridoma cell culture supernatant + 0.4 mg / mL Rituximab, hybridoma cell culture supernatant + 0.04 mg / mL Rituximab).

[0096] 4.3.10.2 Purification from Mixed Sample (0.2 mg / mL Rituximab) 100 μL of the mixed sample (hybridoma cell culture supernatant + 0.4 mg / mL Rituximab) prepared in 4.3.10.1 was injected onto the HisMA20-EEMA column, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3 / 5, 50 mM CAB), and 3 minutes of stabilization buffer (pH 7, 10 mM PB). The measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0097] 4.3.10.3 Purification from Mixed Sample (0.02 mg / mL Rituximab) One mL of the mixed sample (hybridoma cell culture supernatant + 0.04 mg / mL Rituximab) prepared in 4.3.10.1 was injected onto a HisMA20-EEMA column using a Rheodyne 8125, and binding buffer (pH 7, 10 mM PB) was run for 15 minutes using an LC-20AR. After switching the column to an LC-2030, binding buffer (pH 7, 10 mM PB) was run for 3 minutes, elution buffer (pH 3 / 5, 50 mM CAB) for 6 minutes, and stabilization buffer (pH 7, 10 mM PB) for 3 minutes. The measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25 °C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0098] 4.3.10.4 Characterization of Purified Antibodies Eluted from the HisMA20-EEMA Column In the antibody purification experiments performed in 4.3.10.2 and 4.3.10.3, the peak eluting between 7.0 and 8.0 minutes was collected as the antibody peak. However, for fractions eluted with 50 mM CAB at pH 3, 1 M Tris-HCl buffer at pH 9 (1 / 10 of the collected volume) was added to the collection tube beforehand to neutralize the eluent. The collected fractions were then subjected to SEC analysis. A Tosoh Bioscience TSK gel UP-SW3000 size exclusion column (4.6 mm id × 150 mm, 2 μm) was used for the analysis. The mobile phase was 0.2 M PB (0.05% NaN3) at pH 6.7. The analysis was performed at a flow rate of 0.35 mL / min, column oven temperature of 25°C, sample injection volume of 20 μL, and wavelength of 280 nm.

[0099] 4.3.11 Gradient elution with different washing times using binding buffer 2 μL of 4 mg / mL Rituximab was injected onto a HisMA20-EEMA column, and binding buffer (pH 7, 10 mM PB) was run for 3, 10, and 20 minutes, followed by elution buffer (pH 3, 50 mM CAB) for 6 minutes. Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0100] 4.3.12 Column durability and reusability test 10 μL of the mixed sample (hybridoma cell culture supernatant + 4 mg / mL Rituximab) prepared in 4.3.8.1.3 was injected onto the HisMA20-EEMA column, followed by 3 minutes of binding buffer (pH 7, 10 mM PB), 6 minutes of elution buffer (pH 3 / 5, 50 mM CAB), and 11 minutes of stabilization buffer (pH 7, 10 mM PB). This procedure was repeated 100 times to confirm the change in antibody recovery rate with the number of injections. Measurement conditions were a flow rate of 0.2 mL / min, column oven temperature of 25°C, and wavelength of 280 nm. Correction was performed using background data measured without sample injection.

[0101] Example 2: Antibody isolation 5.1 Preparation of packing material 5.1.1 Ninhydrin reaction The ninhydrin reaction was carried out on TOYOPEARL before and after binding of V-501. The results of measuring the absorbance of TOYOPEARL after the ninhydrin reaction are shown in Figure 2.

[0102] Visual inspection confirmed that the purple color was weaker after V-501 binding than before. Furthermore, the absorbance measurements shown in Figure 2 revealed that the absorbance at 580 nm was reduced by 97% after modification compared to before modification. This is thought to be due to V-501 binding to the terminal amine of TOYOPEARL, reducing the number of amino groups available for reaction with ninhydrin. These results demonstrate that V-501 binding to TOYOPEARL was successful.

[0103] 5.2 HPLC measurements 5.2.1 Examination of the amount of polymer modification Figure 3 shows the elution profile of Rituximab at pH 3 and 7 using HisMA20-tBMA-1 and 2 columns.

[0104] The results in Figure 3(a) show that with beads (HisMA20-tBMA-1) modified with a pH-responsive mixed-charge polymer at a TOYOPEARL / monomer weight ratio of 4 / 1, the antibody was not retained when the mobile phase was pH 7 PB, but was eluted in the same manner as with a pH 3 CAB mobile phase. On the other hand, the results in Figure 3(b) show that with beads (HisMA20-tBMA-2) modified with a pH-responsive mixed-charge polymer at a TOYOPEARL / monomer weight ratio of 1 / 1, the antibody was retained when the mobile phase was pH 7 PB, and was eluted when the mobile phase was pH 3 CAB. This indicates that modifying TOYOPEARL with the same weight of monomer ensures sufficient retention of the antibody in a pH 7 mobile phase. Therefore, in subsequent experiments, we decided to prepare a packing material by modifying TOYOPEARL with the same weight of monomer. Furthermore, when comparing the elution peak intensities at pH 3, the amount of antibody eluted with HisMA20-t-BMA-2 was significantly lower than that with HisMA20-tBMA-1. This is thought to be because the increased modification amount strengthened the hydrophobic interactions between the hydrophobic monomer (tBMA) and the antibody, and the electrostatic interactions between the anionic monomer (MAA) and the antibody, making it more difficult for the antibody to elute.

[0105] 5.2.2 Consideration of slurry solvent The elution behavior of benzene was observed using HisMA20-tBMA-2 columns prepared using three types of slurry solvents. The results are shown in Figure 4. The number of theoretical plates and theoretical plate height for each column are shown in Table 2.

[0106] [Table 2]

[0107] Figure 4 shows that of the three columns, the column packed with MeOH / Chloroform = 1 / 3 (v / v) as the slurry solvent exhibited the sharpest peak. Furthermore, the theoretical plate number and theoretical plate height results shown in Table 2 indicate that the column packed with MeOH / Chloroform = 1 / 3 (v / v) as the slurry solvent exhibited the largest number of theoretical plates and the smallest theoretical plate height. These results demonstrate that MeOH / Chloroform = 1 / 3 (v / v) is the most suitable slurry solvent for column packing. Therefore, in subsequent experiments, columns were prepared using MeOH / Chloroform = 1 / 3 (v / v) as the slurry solvent.

[0108] 5.2.3 Consideration of hydrophobic monomers 5.2.3.1 Basic characteristics evaluation Figure 5 shows the elution profile of Rituximab at pH 3, 4, 5, and 7 using HisMA20-tBMA, EMA, and EEMA columns.

[0109] Both columns retained the antibody when 10 mM PB was used as the mobile phase at pH 7. However, the HisMA20-EMA and EEMA columns eluted the antibody when the salt concentration of PB was increased to 50 and 100 mM (Fig. 5-5 (a)–(c)). This is thought to be because at pH 7, the positively charged antibody adsorbed to the negatively charged packing material, but the increased salt concentration of the mobile phase weakened the ionic bond between the antibody and the stationary phase, allowing the antibody to elute. Since the HisMA20-tBMA column did not elute the antibody at 50 or 100 mM, it is thought that antibody retention is strongest at pH 7. On the other hand, the HisMA20-EEMA column, which eluted the antibody most sharply at pH 7 with 50 and 100 mM PB, is thought to have the weakest antibody retention of the three columns.

[0110] The results of measurements using citrate buffer as the mobile phase showed that the antibody elution peak was sharpest at pH 3 and broadened as the pH approached neutral (Fig. 5-5 (d)-(f)). This is thought to be because the packing surface becomes more positively charged as the pH decreases, making it easier to elute the positively charged antibody due to electrostatic repulsion. Comparing the elution peaks of each column, the HisMA20-tBMA column did not elute the antibody at pH 5 and showed a lower elution peak than the other two columns at pH 3, indicating that it retained the antibody the strongest. On the other hand, the HisMA20-EEMA column showed the sharpest elution peak at all pH CABs, indicating that it retained the antibody the weakest and was the easiest to elute.

[0111] These results indicate that the HisMA20-tBMA, EMA, and EEMA columns all undergo changes in the charge state of the packing surface depending on the environmental pH, and thus function as ion-exchange mode-switching chromatography.

[0112] Next, Figure 6 shows the results for each column when the eluent was pH 3, 10, 50, or 100 mM CAB. The results in Figure 6 indicate that for all columns, the sharpest elution peak was observed when the salt concentration of the eluent was 50 mM. As the salt concentration increases, the ionic bond between the antibody and the packing material weakens, making antibody elution easier. However, the broader peak observed with 100 mM CAB than with 50 mM CAB is likely due to the salting-out effect, which makes antibody elution more difficult than with 50 mM. These results indicate that 50 mM is the optimal buffer concentration for antibody elution, and we therefore decided to use 50 mM CAB as the elution buffer in subsequent experiments.

[0113] 5.2.3.2 Gradient elution Rituximab was eluted using a HisMA20-tBMA, EMA, and EEMA column with a gradient of binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 4 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 7.

[0114] Gradient elution using pH 3 CAB as the eluent demonstrated the sharpest antibody elution for both columns compared to pH 4 and 5 (Fig. 5-7 (a)). Furthermore, for both columns, the elution peak tended to broaden as the eluent pH approached neutral. This is likely due to the fact that the lower the eluent pH, the more positively charged the packing surface becomes, as seen in the results of the basic characterization shown in Fig. 5-5. Comparing the results of gradient elution using pH 5 CAB as the eluent shown in Fig. 5-7 (c), the HisMA20-EEMA column demonstrated the sharpest antibody elution. This is likely due to the stronger hydrophobic interactions between the hydrophobic monomer and antibody when tBMA and EMA are used as the hydrophobic monomers than when EEMA is used, which hinders elution due to electrostatic repulsion.

[0115] Gradient elution was performed five times using a HisMA20-tBMA column with 50 mM CAB at pH 4 as the elution buffer. The results are shown in Figure 8 below.

[0116] Figure 8 shows that when gradient elution was performed using a HisMA20-tBMA column with 50 mM CAB at pH 4 as the elution buffer, poor reproducibility was observed, with peak shapes varying from measurement to measurement. This is thought to be due to the strong hydrophobic interaction between the hydrophobic monomer tBMA and the antibody, which prevented antibody elution due to electrostatic repulsion at pH 4, making stable elution difficult.

[0117] The recovery rates of Rituximab during the gradient elution shown in Figure 7 are shown in Figure 9. However, the recovery rate when using a HisMA2-tBMA column with pH 4, 5 CAB as the elution buffer could not be calculated because antibody elution was irregular or did not occur.

[0118] As can be seen from Figure 9, the HisMA20-EEMA column showed a high antibody recovery rate of 93% or higher in gradient elution using all acidic buffers. In particular, an antibody recovery rate of 95% was observed even when CAB at pH 5, the closest to neutral, was used, demonstrating that gradient elution of Rituximab using 50 mM CAB at pH 5 as the elution buffer is possible with the HisMA20-EEMA column.

[0119] 5.2.4 Retention Capacity Test Antibody retention capacity tests were performed using a HisMA20-EEMA column. 10 μL of antibody sample (2.5, 5.0, 10 mg / mL) was injected, followed by binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 4 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 10.

[0120] As shown in Figure 10, when 10 μL of a 2.5 or 5.0 mg / mL antibody sample was injected and 25 or 50 μg of Rituximab was injected at once, no elution of Rituximab was observed with the binding buffer (pH 7, 10 mM PB). Furthermore, when 10 μL of a 10 mg / mL antibody sample was injected and 100 μg of Rituximab was injected at once, a slight elution of the antibody with the binding buffer was observed, but the result did not significantly exceed the column's retention capacity.

[0121] 5.2.5 Gradient elution of mouse IgG Gradient elution of mouse IgG was performed using a HisMA20-EEMA column. 10 μL of 1 mg / mL mouse IgG was injected, followed by binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 4 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 11.

[0122] Figure 11 shows that mouse IgG was eluted by the binding buffer (pH 7, 10 mM PB). Furthermore, a small amount of mouse IgG continued to elute even after switching the mobile phase to the elution buffer (pH 3, 50 mM CAB). This suggests that some of the injected mouse IgG was retained by the binding buffer and then eluted by the elution buffer. These results demonstrate that the HisMA20-EEMA column cannot adequately retain mouse IgG at pH 7.

[0123] 5.2.6 Purification of Rituximab from Mixed Samples 5.2.6.1 Purification of Antibodies from Mixtures with Bovine Serum Albumin (BSA) A purification experiment of Rituximab from a mixture of Rituximab and BSA was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 12.

[0124] Comparing the results in Figure 12 with those in Figure 7, in addition to the antibody peak eluted by the pH 3 and 5 CAB, a peak eluted around 1 minute by the pH 7 PB run first can be seen in Figure 10. The pI of BSA, contained as a contaminant in the mixed sample, is 4.7, and it is negatively charged at pH 7. Therefore, it is thought that electrostatic repulsion occurs between BSA and the similarly negatively charged packing material at pH 7, causing it to be eluted from the column.

[0125] In the purification experiment of FIG. 12, the fractions obtained by separating the elution peak were subjected to SEC analysis, and the results are shown in FIG.

[0126] As shown in Figure 13, the retention times of the standard sample were 4.8 min for BSA and 4.4 min for Rituximab. The mixed sample before separation also showed peaks at 4.4 min and 4.8 min. Furthermore, the retention time of fraction 1 collected in the purification experiment shown in Figure 12 was 4.8 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, eluted first with pH 7 PB, contains only the contaminant BSA, and that fraction 2, eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only Rituximab. Therefore, purification of Rituximab from the mixed sample with BSA was achieved.

[0127] 5.2.6.2 Purification of antibodies from mouse ascites mixtures A purification experiment of Rituximab from a mixed sample of Rituximab and mouse ascites was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min), in that order. The results are shown in Figure 14.

[0128] Similar to the results of the purification experiment from the mixed sample with BSA shown in Figure 12, in addition to the antibody peak eluted by the pH 3, 5 CAB, a peak eluted by the first run, pH 7 PB, was confirmed. It is thought that when the mixed sample of mouse ascites and Rituximab was injected onto the HisMA20-EEMA column, only the antibody was retained on the column by pH 7 PB, and then the antibody retained by the pH 3, 5 CAB was eluted.

[0129] In the purification experiment of FIG. 14, the fractions obtained by separating the elution peak were subjected to SEC analysis, and the results are shown in FIG.

[0130] As shown in Figure 15, the retention times of the standard sample were 4.9 min for mouse ascites and 4.4 min for Rituximab. Peaks at 4.4 min and 4.9 min were also observed in the mixed sample before separation. Furthermore, in the purification experiment shown in Figure 5-14, the retention time of fraction 1 was 4.9 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, eluted first with pH 7 PB, contains only the contaminant mouse ascites, and fraction 2, eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only Rituximab. Therefore, purification of Rituximab from the mixed sample with mouse ascites was achieved.

[0131] 5.2.6.3 Purification of antibodies from hybridoma cell culture supernatant mixtures A purification experiment of Rituximab from a mixture of Rituximab and hybridoma cell culture medium was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 16.

[0132] Similar to the results of the separation experiments using BSA and mouse ascites fluid as impurities, a peak eluted around 1 minute by PB at pH 7 was confirmed in Fig. 5-16. Even when a sample containing a mixture of antibody and hybridoma cell culture supernatant was injected into the HisMA20-EEMA column, it is thought that only the antibody was retained by PB at pH 7, and then the antibody retained by CAB at pH 3 and 5 was eluted.

[0133] In the purification experiment of FIG. 16, the fractions obtained by separating the elution peak were subjected to SEC analysis, and the results are shown in FIG.

[0134] As shown in Figure 17, the retention times of the standard sample were 4.8 min for hybridoma cell culture supernatant and 4.4 min for Rituximab. Peaks at 4.4 min and 4.8 min were also observed in the mixed sample before separation. Furthermore, in the purification experiment shown in Figures 5-16, the retention time of fraction 1 was 4.8 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, eluted first with pH 7 PB, contains only the contaminant hybridoma cell culture supernatant, and fraction 2, eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only Rituximab. Therefore, purification of Rituximab from the mixed sample with hybridoma cell culture supernatant was achieved.

[0135] Figure 18 shows the antibody recovery rates in the purification experiments from the mixed samples shown in Figures 12, 14, and 16. The antibody recovery rates were calculated based on the peak area when 10 μL of 2 mg / mL Rituximab was injected without a column.

[0136] Figure 18 shows that in antibody purification experiments using BSA and mouse ascites fluid as contaminants, nearly 100% of the antibody was recovered. Furthermore, even in purification experiments using hybridoma cell culture supernatant as a contaminant, which is the condition closest to the actual sample, a high antibody recovery rate of over 80% was achieved.

[0137] 5.2.7 Purification of Trastuzumab from Mixed Samples 5.2.7.1 Purification of Antibodies from Mixtures with Bovine Serum Albumin (BSA) A purification experiment of Trastuzumab from a mixture of Trastuzumab and BSA was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min), in that order. The results are shown in Figure 19.

[0138] The results in Figure 19 show that in addition to the antibody peaks eluted by the pH 3 and 5 CAB, a peak around 1 minute eluted by the first run, pH 7 PB, can be seen. The pI of BSA, contained in the mixed sample as a contaminant, is 4.7, and it is negatively charged at pH 7. Therefore, it is thought that electrostatic repulsion occurs between BSA and the negatively charged packing material at pH 7, causing it to be eluted from the column.

[0139] In the purification experiment of FIG. 19, the results of SEC analysis of the fractions collected from the elution peak are shown in FIG.

[0140] As shown in Figure 20, the retention times of the standard sample were 4.8 min for BSA and 4.4 min for Trastuzumab. The mixed sample before separation also showed peaks at 4.4 min and 4.8 min. Furthermore, in the purification experiment shown in Figure 5-19, the retention time of fraction 1 was 4.8 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, which elutes first with pH 7 PB, contains only the contaminant BSA, and fraction 2, which is eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only Trastuzumab. Therefore, purification of Trastuzumab from the mixed sample with BSA was achieved.

[0141] 5.2.7.2 Purification of antibodies from mouse ascites mixtures A purification experiment of Trastuzumab from a mixed sample of Trastuzumab and mouse ascites was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min), in that order. The results are shown in Figure 21.

[0142] Similar to the results of the purification experiment from the mixed sample with BSA shown in Figure 19, in addition to the antibody peak eluted by the pH 3, 5 CAB, a peak eluted by the first loaded pH 7 PB was confirmed. It is thought that when the mixed sample of mouse ascites and Trastuzumab was injected onto the HisMA20-EEMA column, only the antibody was retained on the column by the pH 7 PB, and then the antibody retained by the pH 3, 5 CAB was eluted.

[0143] In the purification experiment of FIG. 21, the results of SEC analysis of the fractions collected from the elution peak are shown in FIG. 22 below.

[0144] As shown in Figure 22, the retention times of the standard sample were 4.9 min for mouse ascites and 4.4 min for Trastuzumab. Peaks at 4.4 min and 4.9 min were also observed in the mixed sample before separation. Furthermore, in the purification experiment shown in Figure 5-21, the retention time of fraction 1 was 4.9 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, eluted first with pH 7 PB, contains only the contaminant mouse ascites, and fraction 2, eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only trastuzumab. Therefore, purification of trastuzumab from the mixed sample with mouse ascites was achieved.

[0145] 5.2.7.3 Purification of antibodies from hybridoma cell culture supernatant mixtures A HisMA20-EEMA column was used to purify Trastuzumab from a mixture of Trastuzumab and hybridoma cell culture fluid. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results are shown in Figure 23.

[0146] Similar to the results of the separation experiments using BSA and mouse ascites fluid as impurities, a peak eluted around 1 minute by PB at pH 7 was confirmed in Fig. 5-23. Even when a sample containing a mixture of antibody and hybridoma cell culture supernatant was injected into the HisMA20-EEMA column, it is thought that only the antibody was retained by PB at pH 7, and then the antibody retained by CAB at pH 3 and 5 was eluted.

[0147] In the purification experiment of FIG. 23, the results of SEC analysis of the fractions collected from the elution peak are shown in FIG.

[0148] As shown in Figure 24, the retention times of the standard sample were 4.8 min for hybridoma cell culture supernatant and 4.4 min for Trastuzumab. Peaks at 4.4 min and 4.8 min were also observed in the mixed sample before separation. Furthermore, in the purification experiment shown in Figure 5-23, the retention time of fraction 1 was 4.8 min, and the retention time of fraction 2 was 4.4 min regardless of the pH elution. This confirms that fraction 1, eluted first with pH 7 PB, contains only the hybridoma cell culture supernatant contaminant, and fraction 2, eluted by subsequently switching the mobile phase to pH 3 / 5 CAB, contains only Trastuzumab. Therefore, purification of Trastuzumab from the mixed sample with hybridoma cell culture supernatant was achieved.

[0149] Fig. 5-25 shows the antibody recovery rates in the purification experiments from the mixed samples shown in Fig. 5-19, 21, and 23. The antibody recovery rates were calculated based on the peak area when 10 μL of 2 mg / mL Trastuzumab was injected without a column.

[0150] Figure 25 shows that in antibody purification experiments using BSA and mouse ascites fluid as contaminants, more than 90% of the antibody was recovered. Furthermore, even in purification experiments using hybridoma cell culture supernatant as a contaminant, which is the condition closest to the actual sample, a high antibody recovery rate of more than 80% was achieved.

[0151] 5.2.8 Antibody purification from low-concentration samples A purification experiment of Rituximab from a mixed sample (hybridoma cell culture supernatant + 0.4 or 0.04 mg / mL Rituximab) was performed using a HisMA20-EEMA column. The eluents were binding buffer (pH 7, 10 mM PB, 3 min), elution buffer (pH 3 / 5, 50 mM CAB, 6 min), and stabilization buffer (pH 7, 10 mM PB, 3 min). The results of each measurement were compared with those obtained in 5.2.6.3 (hybridoma cell culture supernatant + 4 mg / mL Rituximab) and are shown in Figure 26.

[0152] The recovery rates of Rituximab in each measurement compared in FIG. 26 are shown in FIG.

[0153] These results demonstrated that even when the concentration of Rituximab was 0.02 mg / mL, it was possible to capture and purify 80% or more of Rituximab from the hybridoma culture supernatant.

[0154] Furthermore, the results of SEC analysis confirmed that the elution peak contained only Rituximab, and that no denaturation or aggregate formation had occurred during purification (FIG. 28).

[0155] 5.2.8 Gradient elution with different washing times using binding buffer The Rituximab sample was injected into the HisMA20-EEMA column, and the binding buffer (pH 7, 10 mM PB, 3 min) was run for 3, 10, and 20 min. The mobile phase was then switched to the elution buffer (pH 3, 50 mM CAB), and the results are shown in Figure 29.

[0156] The results in Figure 29 show that under all conditions, the antibody injected into the column was retained by the binding buffer and then by the elution buffer. These results indicate that the retention of the antibody by the binding buffer is not due to the effect of size exclusion.

[0157] 5.2.9 Column durability and reusability test The purification procedure of Rituximab from hybridoma culture supernatant using a HisMA20-EEMA column was repeated 100 times, and the results are shown in FIG.

[0158] The results shown in Figure 30(a) show that there was no significant change in the antibody retention time or elution peak shape even after 100 injections of antibody from a mixed sample using the HisMA20-EEMA column. Furthermore, the results shown in Figure 30(b) show that there was no decrease in antibody recovery (90% ± 1.6%) even after 100 injections, demonstrating high reproducibility.

[0159] Example 3 Isolation of Fab Fragments method 1. Preparation of antibody fragment samples (1) Antibody fragmentation by papain 5 mg of antibody and 0.25 mL of immobilized papain were added to 20 mM pH 7.0 phosphate buffer (PB) containing L-cysteine ​​(20 mM) and EDTA (10 mM) to a total volume of 1.0 mL. The mixture was shaken (1200 rpm) at 37°C for 18 hours. After the reaction, 0.75 mL of 10 mM pH 7.5 Tris-HCl buffer was added, and the mixture was centrifuged to collect the supernatant. The digested solution was analyzed using a Tosoh TSKgel UP-SW3000 (4.6 mm x 15 cm) column. The measurement conditions were a flow rate of 0.35 mL / min, a mobile phase of 0.2 M pH 6.7 PB (0.05% NaN3), a temperature of 25°C, an injection volume of 5 μL, and a detection wavelength of 280 nm. The antibody digestion rate was calculated from the digested sample measurement results using the following equation (1):

[0160]

number

[0161] (2) Separation of antibody fragments using protein A-agarose The digested antibody solution (0.1 mL), Protein A-agarose (0.1 mL), and 10 mM pH 7.0 PB (binding buffer, 0.1 mL) were mixed and shaken (1500 rpm) at 4°C for 18 hours. After centrifugation and collection of the supernatant, the Protein A-agarose was washed three times with binding buffer (0.3 mL). Then, 50 mM pH 3.0 citrate buffer (CAB, elution buffer, 0.25 mL) was added, and the mixture was shaken (1500 rpm) for 30 minutes, followed by centrifugation and collection of the supernatant. The collected binding and elution buffers were analyzed using a MAbPac RP (4 μL, 3.0 × 100 mm) column. The measurement conditions were: flow rate 1.0 mL / min, mobile phase 0-1 min: water (0.1% TFA) / acetonitrile (0.1% TFA) (80 / 20, v / v), 1-11 min: water (0.1% TFA) / acetonitrile (0.1% TFA) (80 / 20-50 / 50, v / v), 12-13 min: water (0.1% TFA) / acetonitrile (0.1% TFA) (50 / 50-80 / 20, v / v), 13-15 min: water (0.1% TFA) / acetonitrile (0.1% TFA) (80 / 20, v / v), temperature 80 °C, injection volume 20 μL, and detection wavelength 280 nm.

[0162] 2.HPLC analysis The performance of the HisMA20-EEMA column was evaluated using an HPLC system. Samples were placed in LC vials and used. The mobile phase was degassed using ultrasonic waves before use. (1) Calculation of maximum binding capacity Rituximab (10 mg / mL, 10 μL) was continuously injected onto the HisMA20-EEMA column using binding buffer (10 mM pH 7.0 PB) as the mobile phase. After confirming saturation of the antibody elution peak area, the mobile phase was switched to elution buffer (50 mM pH 3.0 CAB) to elute the retained antibody all at once. The measurement conditions were a flow rate of 0.2 mL / min, a temperature of 25°C, and a detection wavelength of 280 nm.

[0163] (2) Evaluation of antibody fragment retention The antibody fragment sample obtained in step 1 was used after exchange with the same buffer as the mobile phase using Amicon Ultra (trade name, 3K, 0.5 mL). All measurements were performed at a column temperature of 25°C and a detection wavelength of 280 nm. The collected sample was analyzed using a MAbPac RP (4 μL, 3.0 × 100 mm) column.

[0164] result 1. Calculation of maximum binding capacity To calculate the maximum antibody binding capacity of the HisMA20-EEMA column, 100 μg of Rituximab (10 mg / mL, 10 μL) was continuously injected with binding buffer (10 mM pH 7.0 PB) until the elution peak area was saturated. The total amount of antibody retained was then calculated from the peak area of ​​the antibody eluted simultaneously with elution buffer (50 mM pH 3.0 CAB) (Figure 31). From the calculated antibody retention amount and the column volume, the maximum binding capacity was calculated to be 5.51 mg / mL.

[0165] 2. Application to antibody fragment separation (1) Preparation of antibody fragment samples (i) Antibody fragmentation by papain Antibodies were fragmented by cleaving the hinge region using immobilized papain in the presence of a reducing agent (L-cysteine) to enhance the enzymatic activity of papain. Papain digestion was evaluated by size exclusion chromatography (SEC), confirming the appearance of peaks derived from Fab and Fc (5.2-5.7 min) in the digested sample (Figure 32). Furthermore, the digestion rate of each antibody was calculated from the area values ​​of the IgG-derived peak (around 4.5 min) and the fragment-derived peaks in the digested sample, confirming that almost all of the antibody was digested (Table 3).

[0166] [Table 3]

[0167] (ii) Separation of antibody fragments using protein A-agarose Fab and Fc were separated and recovered from the antibody solution after digestion with immobilized papain using protein A-agarose. The separation of antibody fragments was evaluated by reverse-phase chromatography. For Rituximab, the binding buffer and elution buffer were confirmed to contain highly purified Fab and Fc (including undigested antibody), respectively (Figure 33(a)). Similarly, for Adalimumab and Belimumab, highly purified Fab was confirmed to be recovered from the binding buffer (Figure 33(b) and (c)).

[0168] (2) Evaluation of antibody fragment retention The prepared antibody fragment samples were injected into a HisMA20-EEMA column, and the relationship between mobile phase pH and elution rate was investigated (Figure 34). Because all three types of antibodies have the Fc region of human IgG1, the Fc obtained by digesting Rituximab was used as the measurement sample, as it was the Fc common to the three types.

[0169] First, at pH 6.5-7.5, the elution rates of Adalimumab and Belimumab Fab fragments tended to decrease with decreasing pH. This is due to the increased positive charge of the Fab fragments as the pH decreased, strengthening the electrostatic interaction with the negatively charged packing surface. On the other hand, the Fc fragments and Rituximab Fab fragments remained negatively and positively charged, respectively, at pH 6.5-7.5, suggesting that pH did not significantly affect their retention. This difference in the retention behavior of antibody fragments is supported by the isoelectric points of each antibody fragment calculated from the amino acid sequence using a Peptide Calculator (Bachem, Peptide Calculator & Amino Acid Calculator, https: / / www.bachem.com / knowledge-center / peptide-calculator / ) (Table 4). Compared to Fab fragments, Fc fragments have a lower isoelectric point, demonstrating their retention on the column at pH 6.0 or below.

[0170] [Table 4]

[0171] In addition, at pH 5.0-6.5, the Fab dissolution rate tended to increase with decreasing pH, which is thought to be due to the surface charge of the packing material changing from negative to positive as the pH decreased, resulting in the Fab dissolution due to electrostatic repulsion.

[0172] These results suggest that Fab fragments are most strongly retained at pH 6.5, while Fc fragments are completely eluted, suggesting that Fab fragments can be efficiently separated by using 10 mM pH 6.5 PB as the binding buffer.

[0173] (3) Purification of Fab from digested samples Fab was purified from the digested antibody sample using gradient elution, switching the mobile phase pH from 6.5 to 5.0. After sample injection, binding buffer (10 mM pH 6.5 PB) was run for 3 minutes to elute Fc, followed by elution buffer (50 mM pH 5.0 CAB) for 6 minutes to elute Fab (Figure 35(a)-(c)). Comparison of the peak areas eluted by elution buffer confirmed that Rituximab, Adalimumab, and Belimumab were the largest in this order. Each elution peak was collected and the purity of the separated fragments was evaluated by reversed-phase chromatography (Figure 35(d)-(f)). Neither binding buffer showed a Fab-derived peak, confirming that all Fab in the digested sample was retained on the column. The elution buffer showed no Fc-derived peak, and only a Fab-derived peak was observed, demonstrating successful purification of highly pure Fab.

Claims

1. The following general formula [I] 【Chemistry 1】 (In the formula, R 1 , R 2 , R 3 and R 4 are, independently of each other, organic groups constituting the main chain, R 5 is a pH-responsive cationic group containing an imidazole skeleton, R 6 is a pH-responsive anionic group, R 7 is an uncharged organic group, R 8 represents a crosslinkable group, l represents an integer of 1 or more, m represents an integer of 1 or more, n represents an integer of 0 or more, and o represents an integer of 0 or more. R 1 ~R 8 may be of a single type or of multiple types) It is a pH-responsive copolymer whose charge changes depending on the pH.

2. The cationic group is represented by the following general formula: 【Chemistry 2】 (In each formula, R 9 is an optional spacer group, R 10 ~R 12 each independently represents hydrogen or an organic group.

2. The pH-responsive copolymer according to claim 1, which is at least one selected from the group consisting of:

3. The pH-responsive copolymer of claim 2 , wherein the cationic group is a histamine methacrylamide residue.

4. The anionic group is represented by the following general formula: 【Transformation 3】 (In the formula, R 13 is an optional spacer group, R 14 ~R 19 each independently represents hydrogen or an organic group.

2. The pH-responsive copolymer according to claim 1, which is at least one selected from the group consisting of:

5. The pH-responsive copolymer according to claim 4 , wherein the anionic group is a group containing a carboxyl group.

6. R 7 The pH-responsive copolymer according to claim 1, wherein is a methacrylic acid ester residue or an acrylic acid ester residue.

7. R 1 , R 2 , R 3 and R 4 and each independently represent an alkyl group having 2 to 4 carbon atoms.

8. The pH-responsive copolymer according to claim 1 , wherein the crosslinkable group is a group containing an ethylenically unsaturated bond.

9. The pH-responsive copolymer according to any one of claims 1 to 8, wherein a plurality of molecules are crosslinked by crosslinking between the crosslinkable groups.

10. R 1 If there are 100 moles of units containing R 2 20 to 500 moles of units containing R 3 The unit containing R is 0 to 2000 moles. 4 2. The pH-responsive copolymer according to claim 1, wherein the number of units comprising: is 0 to 400 moles.

11. The pH-responsive copolymer according to any one of claims 1 to 10, wherein one end is bound to a particle.

12. A column packing material comprising the pH-responsive copolymer according to claim 11.

13. The column packing material according to claim 12, which is a column packing material for antibody separation.

14. A method for separating antibodies, comprising the steps of: applying a sample solution containing antibodies to the antibody separation column packing material described in claim 13 packed in a column; then applying a binding buffer to the column to remove impurities in the sample solution; and then applying an elution buffer to the column to elute the antibodies in the sample solution.

15. The column packing material according to claim 12, which is a column packing material for separating antigen-binding fragments of antibodies.

16. A method for separating antigen-binding fragments of antibodies, comprising the steps of: applying a sample solution containing antigen-binding fragments of antibodies to a column packing material for separating antigen-binding fragments of antibodies according to claim 15 packed in a column; then applying a binding buffer to the column to remove contaminants in the sample solution; and then applying an elution buffer to the column to elute the antigen-binding fragments of antibodies in the sample solution, wherein the pH of the binding buffer is such that a majority of the antigen-binding fragments of interest remain bound to the column packing material.

17. 17. The method of claim 16, wherein the pH of the binding buffer is between 6.0 and 7.

5.

18. The method of claim 17, wherein the antigen-binding fragment is a Fab fragment.

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