JP2023174125A - METHOD FOR PURIFYING ANTIBODY USING Fc-BINDING PROTEIN IMMOBILIZED ADSORBENT - Google Patents

Abstract

To provide a method for efficiently purifying an antibody included in a sample by using an antibody adsorbent that includes an insoluble carrier and a human neonatal Fc receptor immobilized to the carrier.SOLUTION: The foregoing problem is solved by adjusting the pH levels of an equilibration liquid, used for equilibrating a column filled with the adsorbent, and the sample to be 4.5 or more and 5.7 or less.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method for purifying antibodies contained in a sample using an adsorbent containing an insoluble carrier and an Fc-binding protein immobilized on the carrier. In particular, the present invention relates to a highly efficient method for purifying the antibody using an adsorbent containing an insoluble carrier and a human neonatal Fc receptor immobilized on the carrier.

Fc receptors are receptor proteins that bind to the Fc region of immunoglobulin (antibody) molecules, and bind to immune complexes of antigens and immunoglobulins to transmit signals within cells (Non-Patent Document 1). Examples of Fc receptors include Fcγ receptor, Fcα receptor, Fcε receptor, which belong to the immunoglobulin superfamily, and neonatal Fc receptor (FcRn), which is a major histocompatibility complex (MHC) class I-related molecule. It will be done.

As mentioned above, Fc receptors are proteins that bind to the Fc region of immunoglobulin molecules (hereinafter also referred to as "Fc-binding proteins"). Therefore, an adsorbent obtained by immobilizing Fc receptors on an insoluble carrier can purify immunoglobulins (antibodies) contained in a sample to a high degree of purity. Patent Document 1 discloses an example of antibody purification using human Fcγ receptor as the Fc receptor, and Patent Document 2 discloses an example of antibody purification using human FcRn as the Fc receptor.

In Patent Document 2, a solution containing an antibody (pH 5.8) is added to a column filled with an adsorbent immobilized with human FcRn that has been equilibrated in advance with a phosphate buffer (pH 5.8), and the antibody is After adsorption to the adsorbent, the antibody adsorbed to the adsorbent is eluted by pH gradient elution using a phosphate buffer (pH 8.0). However, the method described in Patent Document 2 has a low antibody yield, and a more efficient antibody purification method has been sought.

Japanese Patent Application Publication No. 2012-072091 Japanese Patent Application Publication No. 2018-183087

Takai T. , Jpn. J. Clin. Immunol. , 28, 318-326, 2005

An object of the present invention is to provide a method for highly efficiently purifying antibodies contained in a sample using an adsorbent containing an insoluble carrier and a human neonatal Fc receptor immobilized on the carrier.

The present inventors conducted intensive studies to solve the above problems, and as a result, optimized the pH of the equilibration solution of the column packed with the adsorbent immobilized human neonatal Fc receptor (FcRn) and the solution containing the antibody. By doing so, we realized highly efficient antibody purification and completed the present invention.

That is, the present invention includes the embodiments described in [1] to [3] below.

[1] Equilibrating a column packed with an antibody adsorbent containing an insoluble carrier and an Fc-binding protein immobilized on the carrier with an equilibration solution, and applying a solution containing the antibody to the column equilibrated in the above step. A method for purifying an antibody, the method comprising the steps of: adding and adsorbing the antibody to the adsorbent; and eluting the antibody adsorbed to the adsorbent with an eluent, wherein the Fc-binding protein is a human neonatal Fc receptor. (FcRn), and the pH of the equilibration solution and the solution containing the antibody is 4.5 or more and 5.7 or less.

[2] The purification method according to [1], wherein the human FcRn is selected from any of the following (i) to (iii);
(i) Amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1 and from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2. (ii) an amino acid residue from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1 and from the 21st isoleucine in the amino acid sequence set forth in SEQ ID NO: 2; Containing at least the amino acid residues up to the 119th methionine, any one of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in these amino acid residues polypeptide (iii) which has an antibody-binding activity and has an antibody-binding activity, from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1, and the 21st alanine in the amino acid sequence set forth in SEQ ID NO: 2. A polypeptide that has 70% or more homology to the entire amino acid sequence from isoleucine to methionine 119 and has antibody binding activity.

[3] The purification method according to [2], wherein the human FcRn is selected from any of the following (iv) to (vi);
(iv) A polypeptide containing at least the amino acid residues from the 29th alanine to the 426th methionine in the amino acid sequence set forth in SEQ ID NO: 3. (v) From the 29th alanine to the 426th methionine in the amino acid sequence set forth in SEQ ID NO: 3. Contains at least the amino acid residues up to the 426th methionine, but among the amino acid residues, from the 29th alanine to the 302nd serine, and/or from the 328th isoleucine to the 426th methionine, A polypeptide (vi) in SEQ ID NO: 3 that has one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions and has antibody binding activity. Contains at least the amino acid residues from the 29th alanine to the 426th methionine in the amino acid sequence described, provided that the amino acid residues from the 29th alanine to the 302nd serine, and from the 328th isoleucine to the 426th amino acid residue A polypeptide that has 70% homology to the entire amino acid sequence up to methionine and has antibody binding activity.

The present invention comprises a step of equilibrating a column packed with an antibody adsorbent containing an insoluble carrier and human FcRn immobilized on the carrier with an equilibration solution, and adding a solution containing the antibody to the column equilibrated in the step. In the antibody purification method, which includes the steps of adsorbing the antibody to the adsorbent, and eluting the antibody adsorbed to the adsorbent with an eluent, the pH of the equilibration solution and the solution containing the antibody is set to 4. It is characterized by having a value of .5 or more and 5.7 or less. Since the present invention can efficiently purify antibodies contained in a sample, it can be said to be a method suitable for industrial production of antibodies.

This is a chromatogram in which a monoclonal antibody was separated using a column packed with an adsorbent immobilized with Fc-binding protein FcRn-m10ΔW87 (SEQ ID NO: 4). Note that the pH of the equilibration solution of the column and the solution containing the monoclonal antibody is 4.7 (thin solid line), 5.0 (dotted chain line), 5.3 (dotted line), or 5.5 (thick solid line). It is. This is a chromatogram in which a monoclonal antibody was separated using a column packed with an adsorbent immobilized with the Fc-binding protein FcRn-m10ΔW87 (SEQ ID NO: 4). The pH of the column equilibration solution and the solution containing the monoclonal antibody is 5.5 (thick solid line), 5.8 (two-dot chain line), or 6.0 (dashed line).

The present invention will be explained in detail below.

The Fc binding protein in the present invention is
(I) human FcRn that has binding properties to the Fc region of antibodies;
(II) any one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in the amino acid residues of the human FcRn, and the binding property (hereinafter also simply referred to as "antibody binding activity"); and (III) a polypeptide having 70% or more homology to the entire amino acid sequence of human FcRn and having antibody binding activity;
Either.

As one aspect of the Fc-binding protein of the present invention,
(A) Amino acid residues from the 24th alanine (Ala) to the 297th serine (Ser), which corresponds to the extracellular region of the human FcRnα chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 (UniProt No. P55899). base and
(B) Amino acids from the 21st isoleucine (Ile) to the 119th methionine (Met), which correspond to the β2 microglobulin region of the human FcRn β chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 (UniProt No. P61769). Examples include polypeptides containing at least the following residues.

In addition, in the one aspect, it is sufficient that the amino acid residues of (A) and (B) are included at least, and all or part of the signal peptide region on the N-terminal side of (A) and (B) is included. It may contain all or part of the cell membrane-spanning region and extracellular region on the C-terminal side of (A). Further, in the above embodiment, the order of (A) and (B) does not matter. That is, the above (B) may be located on the N-terminal side or the C-terminal side of the above (A). Alternatively, the above (A) and the above (B) may be directly linked, and the above (A) and the above (B) may be combined. As a specific example of human FcRn in which (A) and (B) are linked via a linker, from the 29th alanine (Ala) to the 426th methionine (Met) in the amino acid sequence set forth in SEQ ID NO: 3. Examples include polypeptides containing at least the following amino acid residues. In addition, in the polypeptide, the amino acid residues from the 29th alanine (Ala) to the 302nd serine (Ser) are the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1. The amino acid residues from the 303rd glycine (Gly) to the 327th serine (Ser) are the GS linker, and the 328th isoleucine (Ile) to the 426th methionine (Met) are SEQ ID NO: 2. Each corresponds to the amino acid residues from the 21st isoleucine to the 119th methionine in the described amino acid sequence.

The term "one or several" in the above (II) varies depending on the position of amino acid substitution in the three-dimensional structure of human FcRn and the type of amino acid residue, but examples include 1 to 50, and 1 to 30. 1 to 20 pieces, 1 to 10 pieces, 1 to 9 pieces, 1 to 8 pieces, 1 to 7 pieces, 1 to 6 pieces, 1 to 5 pieces Hereinafter, it means any one of 1 to 4, 1 to 3, 1 to 2, and 1. Examples of "substitution, deletion, insertion, and addition of one or several amino acid residues" include JP 2018-183087A, JP 2020-028285A, JP 2021-073877A, JP 2021-A Examples include substitutions and deletions of amino acid residues disclosed in JP-A-136967 and JP-A-2022-076998. Furthermore, as long as it has antibody binding activity, substitutions, deletions, insertions, and additions of amino acid residues may occur at positions other than the above-mentioned substitutions and deletions of amino acid residues.

Note that the "substitution of one or several amino acid residues" in (II) above includes not only amino acid substitutions at specific positions as described above, but also substitutions between amino acids with similar physical and/or chemical properties. Conservative substitutions may occur. It is known by those skilled in the art that conservative substitution generally maintains the function of the protein between the substitution and the non-substitution. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International Inc., 9, 2005). In addition, "any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues" in (2) above includes naturally occurring mutations (mutants or variants). .

The homology of the amino acid sequence in the above (III) may be 70% or more, and may have higher homology (for example, 80% or more, 85% or more, 90% or more, or 95% or more). Note that in this specification, "homology" may mean similarity or identity, and particularly may mean identity. "Amino acid sequence homology" means homology to the entire amino acid sequence. "Identity" between amino acid sequences refers to the ratio of amino acid residues of the same type in those amino acid sequences (Jikken Igaku, 31(3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type and the ratio of amino acid residues of similar side chain properties in those amino acid sequences (Experimental Medicine, 31 (3) ), Yodosha). Amino acid sequence homology can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

In the present invention, the Fc-binding protein (human FcRn) may further have an oligopeptide (tag peptide) added to its N-terminus or C-terminus, which is useful when it is separated from a solution in the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and the like. Furthermore, a cysteine-containing oligopeptide, which is useful when immobilizing an Fc-binding protein (human FcRn) on an insoluble carrier such as a support for chromatography, is added to the N-terminus or C-terminus of the Fc-binding protein. Further additions may be made. The length of the oligopeptide added to the N-terminus or C-terminus of the Fc-binding protein is not particularly limited as long as it does not impair the antibody-binding activity or stability of the Fc-binding protein. Furthermore, a signal peptide may be added to the N-terminus of the Fc-binding protein to promote efficient expression in the host. Examples of the signal peptide when the host is E. coli include PelB, DsbA, MalE (region from 1 to 26 of the amino acid sequence described in UniProt No. P0AEX9), TorT, etc., which secrete proteins into the periplasm. An example is a signal peptide (Japanese Unexamined Patent Publication No. 2011-097898). A specific example of human FcRn with a signal peptide added to the N-terminus and a tag peptide added to the C-terminus is a polypeptide consisting of the sequence shown in SEQ ID NO: 3. In the polypeptide, the amino acid residues from the 1st methionine (Met) to the 26th alanine (Ala) are the MalE signal peptide (the 1st to 26th amino acid residues of the amino acid sequence described in UniProt No. P0AEX9). 27th methionine (Met) and 28th glycine (Gly) are linkers, and the amino acid residues from 29th alanine (Ala) to 426th methionine (Met) are the aforementioned GS linker. It is human FcRn bound via , glycine (Gly) at positions 427 and 428 is a linker, and histidine (His) from positions 429 to 434 is a polyhistidine tag.

The antibody adsorbent used in the purification method of the present invention may be produced, for example, by binding (immobilizing) the above-described Fc-binding protein to an insoluble carrier. The insoluble carrier is not particularly limited, and includes carriers made from polysaccharides such as agarose, alginate, carrageenan, chitin, cellulose, dextrin, dextran, and starch, polyvinyl alcohol, polymethacrylate, and poly(2- Examples include carriers made from synthetic polymers such as hydroxyethyl methacrylate) and polyurethane, and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels into which hydroxyl groups have been introduced, such as Toyopearl (manufactured by Tosoh Corporation), agarose gels, such as Sepharose (manufactured by Cytiva Corporation), and cellulose gels, such as Cellufine (manufactured by JNC Corporation). There are no particular limitations on the shape of the insoluble carrier, and it may be granular or non-granular, porous or non-porous.

To immobilize an Fc-binding protein on an insoluble carrier, the insoluble carrier may be immobilized with an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetyl group. The Fc-binding protein may be immobilized by providing an active group such as acetamide (iodoacetamide, bromoacetamide, etc.) and covalently bonding the Fc-binding protein to an insoluble carrier via the active group. The carrier provided with an active group may be a commercially available carrier as it is, or may be prepared by introducing an active group onto the surface of the carrier under appropriate reaction conditions. Commercially available carriers with active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, and NHS-activated Sepharo. se 4 Fast Flow, Epoxy-activated Sepharose 6B (both manufactured by Cytiva) and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

On the other hand, as a method for introducing active groups onto the surface of a carrier, there is a method in which one of the compounds having two or more active sites is reacted with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the surface of the carrier. I can give an example. Examples of such compounds include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether as compounds that introduce epoxy groups into hydroxy groups or amino groups on the surface of the carrier. . Examples of compounds that introduce carboxyl groups onto the carrier surface after introducing an epoxy group onto the carrier surface using the above compound include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, and 3-mercaptoacetic acid. Examples include aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

Compounds that introduce maleimide groups into hydroxy groups, epoxy groups, carboxy groups, and amino groups present on the surface of the carrier include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, and 4- (4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide , 4-(maleimido)phenylisocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N -Hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-6-aminohexanoic acid, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl) N-hydroxysuccinimide An example is ester, (m-maleimidobenzoyl) N-hydroxysuccinimide ester.

Compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the carrier surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic bromide, chloroacetic anhydride, and bromoacetic anhydride. , iodoacetic anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester can be exemplified. An example of such a method is to react the hydroxy group or amino group present on the surface of the carrier with ω-alkenyl alkane glycidyl ether, and then halogenate and activate the ω-alkenyl moiety with a halogenating agent. Examples of the ω-alkenyl alkane glycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. can.

Another example of a method for introducing an active group onto the surface of a carrier is a method of introducing an activated group into a carboxy group present on the surface of the carrier using a condensing agent and an additive. Examples of the condensing agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

Buffers used to immobilize Fc-binding proteins on insoluble carriers include acetate buffer, phosphate buffer, MES (2-Morphorinoethanesulfonic acid) buffer, HEPES (2-[4-(2-Hydroxyethyl)- Examples include 1-piperazinyl]ethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization may be appropriately set within the temperature range from 5°C to 50°C, taking into account the reactivity of the active group and the stability of the Fc-binding protein, preferably from 10°C to 50°C. The temperature range is 35°C.

The purification method of the present invention includes:
a step of equilibrating a column packed with the antibody adsorbent produced by the method described above with an equilibration solution; a step of adding a solution containing an antibody to the column equilibrated in the step and adsorbing the antibody to the adsorbent; and eluting the antibody adsorbed to the adsorbent with an eluent, and the pH of the equilibration solution and the solution containing the antibody is set to 4.5 or more and 5.7 or less. It is preferable to set the pH to 4.8 or more and 5.6 or less because antibodies can be purified more efficiently, and it is preferable to set the pH to 5.4 or more and 5.6 or less because antibodies can be purified even more efficiently. preferable. Further, the solution containing the equilibration solution and the antibody is preferably a buffer solution, and examples thereof include a phosphate buffer solution, an acetate buffer solution, and an MES buffer solution, which have a buffering capacity in the above-mentioned pH range. In the purification method of the present invention, it is preferable that the pH of the equilibration solution and the solution containing the antibody be the same. In this specification, equivalent pH means, for example, that the difference between the pH of the equilibration solution and the pH of the solution containing the antibody is 0.2 or less, 0.1 or less, or 0.05 or less.

The antibody that can be purified by the method of the present invention may be any antibody that contains at least the Fc region of an antibody that has affinity for an Fc-binding protein. Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substituted antibodies thereof, which are commonly used as antibodies for antibody medicines. Also, antibodies with artificially modified structures, such as bispecific antibodies, fusion antibodies of the Fc region of an antibody and other proteins, and complexes of the Fc region of an antibody and a drug (ADC), etc. can also be efficiently purified by the method of the present invention.

In order to elute the antibody adsorbed to the antibody adsorbent, an eluent that can weaken the interaction between the antibody and the ligand (Fc-binding protein) may be added. Specifically, the interaction may be weakened by using a pH change due to a buffer solution, a counter peptide, a temperature change, a salt concentration change, or the like. When eluating the antibody adsorbed to the antibody adsorbent using pH change, use a buffer solution with a higher pH (on the basic side) than the equilibration solution or the solution containing the antibody as the eluent to elute the antibody. Just let it happen. The pH of the eluate is, for example, 6.0 or more and 9.0 or less, preferably 6.5 or more and 8.5 or less, and more preferably 7.0 or more and 8.2 or less. Like the equilibration solution and the antibody-containing solution, the eluate is preferably a buffer solution, such as a phosphate buffer, Tris buffer, HEPES buffer, Tricine buffer, or Bicine buffer, which has a buffering capacity in the above pH range. I can give you some liquid. The pH change may be carried out isocratic in which only the eluent is fed as described above, but it is preferably carried out in a gradient manner in which the ratio of the equilibration liquid and the eluate is continuously changed. . Elution by a pH gradient may be performed by a step gradient in which the ratio of the equilibration solution and the eluate is changed stepwise, or by a linear gradient in which the ratio is changed linearly. It's okay.

Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.

Example 1 Preparation of Fc-binding protein (1) A transformant expressing the Fc-binding protein FcRn-m10ΔW87 (SEQ ID NO: 4) was grown in a 2YT liquid medium containing 50 μg/mL of kanamycin (peptone 16 g/L, yeast extract). 10 g/L, sodium chloride 5 g/L) and cultured with shaking at 37° C. overnight. Note that FcRn-m10ΔW87 is a polypeptide in which the amino acid substitutions or deletions shown below from <I> to <XI> have occurred in the Fc binding protein (human FcRn) set forth in SEQ ID NO: 3.
<I> Cysteine at position 76 in SEQ ID NO: 3 (71st in SEQ ID NO: 1) is replaced with arginine <II> Asparagine at position 83 in SEQ ID NO: 3 (78th in SEQ ID NO: 1) is replaced with aspartic acid <III> Deletion of tryptophan at position 87 of SEQ ID NO: 3 (position 82 in SEQ ID NO: 1) <IV> Replacement of glycine at position 156 of SEQ ID NO: 3 (position 151 in SEQ ID NO: 1) with aspartic acid <V> Substitution of aspartic acid at position 156 of SEQ ID NO: 3 Glutamine at position 172 (position 167 in SEQ ID NO: 1) is replaced with glutamic acid Asparagine at position 196 in SEQ ID NO: 1 is replaced with aspartic acid <VIII> Glutamine at position 237 in SEQ ID NO: 3 (232nd in SEQ ID NO: 1) is replaced with leucine <IX> 300th position in SEQ ID NO: 3 (in SEQ ID NO: 1) Lysine at position 295 (position 295) is replaced with glutamic acid <X> Lysine at position 333 of SEQ ID NO: 3 (position 26 in SEQ ID NO: 2) is replaced with isoleucine <XI> Position 387 of SEQ ID NO: 3 (position 80 in SEQ ID NO: 2) is replaced with isoleucine. Replacement of tryptophan with serine (2) After culturing, 30 mL of the culture solution was subcultured into 1 L of 2YT liquid medium, and cultured with shaking at 37° C. for 2 hours. Thereafter, protein expression was induced with 0.05 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and further cultured with shaking at 20° C. overnight.

(3) After culturing, the medium was removed by centrifugation to collect E. coli.

(4) Add a disruption solution (100mM Tris-HCl (pH 8.0) containing 8M urea) of 5 times the amount of bacterial cells to the obtained Escherichia coli, and use an ultrasonic disruptor (manufactured by Tomy Seiko Co., Ltd.) to disrupt the Escherichia coli. It was crushed. Only the supernatant was collected by centrifugation, and a protein extract of the soluble fraction was prepared.

(5) Apply the protein extract from (4) to a Ni Sepharose (manufactured by Cytiva) column, wash with washing buffer (100mM Tris-HCl (pH 8.0) containing 500mM NaCl and 8M urea), and then add refolding buffer. Refolding was performed with (10mM Tris-HCl (pH 8.0)) and elution was performed with an elution buffer (20mM Tris-HCl (pH 8.0) containing 150mM NaCl and 250mM imidazole).

(6) After confirming the presence of Fc-binding proteins in the elution fraction by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis), concentrate the fraction and add buffer (20mM Tris-HCl containing 150mM NaCl). (pH 8.0)) to remove the imidazole. As a result of calibration using the Bradford method, the amount of crudely purified protein obtained was 24 mg.

Example 2 Immobilization of Fc-binding protein onto an insoluble carrier 2 g of a porous hydrophilic polymer for adsorption material (manufactured by Tosoh Corporation, G5000PW) was collected as a suction dry gel, and the hydroxyl groups on the surface of the gel were converted into iodoacetyl groups. After activation,
By reacting the entire amount (24 mg) of the crudely purified FcRn-m10ΔW87 protein obtained in Example 1, an FcRn-m10ΔW87 immobilized gel was obtained.

Example 3 Antibody purification using an antibody adsorbent packed column (1) 1.25 mL of the FcRn-m10ΔW87 immobilized gel obtained in Example 2 (hereinafter also simply referred to as "antibody adsorbent") was transferred to a φ4.6 mm x 75 mm An antibody adsorbent column was prepared by filling a stainless steel column (manufactured by Tosoh Corporation).

(2) After connecting the antibody adsorbent column prepared in (1) to the Nexera system (manufactured by Shimadzu Corporation), add 50 mM phosphate buffer (hereinafter also referred to as "equilibration solution") to the column at a flow rate of 0.5 mL. The column was equilibrated by pumping 5 column volumes (hereinafter referred to as CV; 1 CV = 1.25 mL) at a rate of /min. Note that the pH of the equilibration solution is any one of 4.7, 5.0, 5.3, 5.5, 5.8, and 6.0.

(3) After diluting monoclonal antibodies (Rituxan manufactured by Zenyaku Kogyo Co., Ltd.) to 1.0 mg/mL with 50 mM phosphate buffer having the same pH as each equilibration solution used in (2), the diluted solution was used for antibody adsorption. The antibody was adsorbed onto the antibody adsorbent by feeding 5 μL (loading 5 μg of antibody) into the antibody column at a flow rate of 0.5 mL/min.

(4) Subsequently, after feeding the equilibration solution to the antibody adsorbent column at a flow rate of 0.5 mL/min for 5 minutes, the eluate (50 mM phosphate buffer (pH 8.0)) was % to the antibody adsorbent column, and the antibody adsorbed to the antibody adsorbent was eluted. After the antibody elution, the buffer solution used in the equilibration in (2) was flowed through the antibody adsorbent column for 10 minutes to equilibrate it.

Chromatograms in which monoclonal antibodies were separated are shown in FIGS. 1 and 2. Figure 1 shows the results when the pH of the equilibration solution and antibody dilution solution was set to 4.7, 5.0, 5.3, and 5.5, and Figure 2 shows the results when the pH was set to 5.5, 5.5, and 5.5. These are the results when either .8 or 6.0 was used.

In the conventional purification method using a human FcRn-immobilized adsorbent (Japanese Patent Application Laid-Open No. 2018-183087), the pH of the equilibration solution and the solution containing the antibody was 5.8, but the pH was adjusted to the acidic side, specifically By adjusting the pH to 4.5 or more and 5.7 or less, the peak height corresponding to a monoclonal antibody can be significantly improved, allowing efficient antibody purification. From the results shown in Figures 1 and 2, it is clear that the pH of the equilibration solution and antibody dilution solution, which have relatively high peak heights corresponding to monoclonal antibodies, is set to 4.8 or more and 5.6 or less. The antibody can be purified even more efficiently when the peak height corresponding to the monoclonal antibody is at its highest at around pH 5.5, specifically at pH 5.4 or higher and 5.6 or lower.

Claims (3)

A step of equilibrating a column packed with an antibody adsorbent containing an insoluble carrier and an Fc-binding protein immobilized on the carrier with an equilibration solution, and adding a solution containing the antibody to the column equilibrated in the above step. A method for purifying an antibody, comprising the steps of adsorbing the antibody to the adsorbent, and eluting the antibody adsorbed to the adsorbent with an eluent, the method comprising:
The purification method, wherein the Fc-binding protein is human neonatal Fc receptor (FcRn), and the pH of the equilibration solution and the solution containing the antibody is 4.5 or more and 5.7 or less. The purification method according to claim 1, wherein the human FcRn is selected from any of the following (i) to (iii);
(i) Amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1 and from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2. (ii) an amino acid residue from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1 and from the 21st isoleucine in the amino acid sequence set forth in SEQ ID NO: 2; Containing at least the amino acid residues up to the 119th methionine, any one of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in these amino acid residues polypeptide (iii) which has an antibody-binding activity and has an antibody-binding activity, from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 1, and the 21st position of the amino acid sequence set forth in SEQ ID NO: 2. A polypeptide that has 70% or more homology to the entire amino acid sequence from isoleucine to methionine 119 and has antibody binding activity. The purification method according to claim 2, wherein the human FcRn is selected from any of the following (iv) to (vi);
(iv) A polypeptide containing at least the amino acid residues from the 29th alanine to the 426th methionine in the amino acid sequence set forth in SEQ ID NO: 3. (v) From the 29th alanine to the 426th methionine in the amino acid sequence set forth in SEQ ID NO: 3. Contains at least the amino acid residues up to the 426th methionine, but among the amino acid residues, from the 29th alanine to the 302nd serine, and/or from the 328th isoleucine to the 426th methionine, A polypeptide (vi) in SEQ ID NO: 3 that has one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions and has antibody binding activity. Contains at least the amino acid residues from the 29th alanine to the 426th methionine in the amino acid sequence described, provided that the amino acid residues from the 29th alanine to the 302nd serine, and from the 328th isoleucine to the 426th amino acid residue A polypeptide that has 70% homology to the entire amino acid sequence up to methionine and has antibody binding activity.