Proteins with immunoglobulin-binding activity

Abstract

The objective is to provide a protein with immunoglobulin-binding activity that can be used as a ligand protein for an immunoglobulin adsorbent, enabling antibody elution under milder pH conditions than conventional immunoglobulin adsorbents. [Solution] A protein comprising the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, wherein the protein has one or more amino acid substitutions selected from (1) and (2) below: (1) The amino acid residue corresponding to the 25th threonine in SEQ ID NO: 1 is replaced with histidine. (2) The amino acid residue corresponding to the 29th lysine residue in Sequence ID No. 1 is replaced with histidine. To provide.

Description

[Technical Field]

[0001] The present disclosure relates to a protein having immunoglobulin binding activity. [Background Art]

[0002] Antibodies are widely used in research reagents, antibody pharmaceuticals, and the like. These reagents and pharmaceutical antibodies are generally purified by affinity chromatography. For antibody affinity purification, a column on which a ligand, which is a substance that specifically binds to an antibody (immunoglobulin), is immobilized is used. Immunoglobulin binding proteins such as Protein A, Protein G, and Protein L are used as this ligand.

[0003] Affinity purification of antibodies generally includes a step of adsorbing the antibody to an affinity carrier under neutral pH conditions, then eluting the antibody from the carrier under acidic pH conditions. Since the acidic pH condition damages antibodies and causes aggregation, elution is preferably performed under mild acidic pH conditions.

[0004] As methods for increasing the pH during elution (that is, bringing it closer to neutrality), a method of inserting an oligopeptide having flexible amino acid residues into an immunoglobulin binding protein (Non-Patent Document 1), and a method of substituting specific amino acid residues of an immunoglobulin binding protein (Patent Document 1, Patent Document 2) are known. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. WO 2019 / 059400 [Patent Document 2] Japanese Unexamined Patent Publication No. 2023-103953 [Non-Patent Documents]

[0006] [Non-Patent Document 1] Susanne Gulich et al., Journal of Biotechnology, 2000, 76, 233-244 [Overview of the project] [Problems that the invention aims to solve]

[0007] This disclosure aims to provide a protein having immunoglobulin-binding activity that can be used as a ligand protein for an immunoglobulin adsorbent that enables antibody elution under milder pH conditions than conventional immunoglobulin adsorbents. [Means for solving the problem]

[0008] The inventors investigated the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia. As a result, the inventors found that the aforementioned problem can be solved by having a specific amino acid substitution in the amino acid sequence.

[0009] In other words, this disclosure includes the following [1] to

[11] . [1] A protein comprising the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, wherein the amino acid sequence has one or more amino acid substitutions selected from (1) and (2) below: (1) The amino acid residue corresponding to the 25th threonine in SEQ ID NO: 1 is replaced with histidine. (2) The amino acid residue corresponding to the 29th lysine residue of Sequence ID No. 1 is replaced with histidine. [2] The protein described in [1] above, selected from (a) or (b): (a) Proteins comprising the amino acid substitutions described in Sequence ID No. 1, including those in (1) above and (3) through (18) below, or those in (2) above and (3) through (8) and (10) through (18) below; (3) The amino acid residue corresponding to the fourth glutamic acid in SEQ ID NO: 1 is replaced with glycine. (4) The amino acid residue corresponding to the 6th proline in SEQ ID NO: 1 is replaced with serine. (5) The amino acid residue corresponding to the 7th lysine residue in SEQ ID NO: 1 is replaced with alanine. (6) The amino acid residue corresponding to the 13th lysine residue in SEQ ID NO: 1 is replaced with arginine. (7) The amino acid residue corresponding to the 22nd lysine residue in SEQ ID NO: 1 is replaced with arginine. (8) The amino acid residue corresponding to isoleucine at position 23 of SEQ ID NO: 1 is replaced with arginine. (9) The amino acid residue corresponding to the 29th lysine residue in Sequence ID No. 1 is replaced with isoleucine. (10) The amino acid residue corresponding to lysine at position 38 of SEQ ID NO: 1 is replaced with glutamic acid. (11) The amino acid residue corresponding to asparagine at position 44 of SEQ ID NO: 1 is replaced with arginine. (12) The amino acid residue corresponding to the 48th lysine residue in Sequence ID No. 1 is replaced with arginine. (13) The amino acid residue corresponding to glutamic acid at position 49 of SEQ ID NO: 1 is replaced with aspartic acid. (14) The amino acid residue corresponding to asparagine at position 50 of SEQ ID NO: 1 is replaced with tyrosine. (15) The amino acid residue corresponding to tyrosine at position 53 of SEQ ID NO: 1 is substituted with phenylalanine. (16) The amino acid residue corresponding to asparagine at position 62 of SEQ ID NO: 1 is replaced with tyrosine. (17) The amino acid residue corresponding to lysine at position 67 of Sequence ID No. 1 is replaced with arginine. (18) The amino acid residue corresponding to alanine at position 69 of sequence number 1 is substituted with valine. (b) A protein comprising an amino acid sequence described in Sequence ID No. 1, wherein the amino acid substitutions described in (1) and (3) to (18), or the amino acid substitutions described in (2) to (8) and (10) to (18), are identical to the entire amino acid sequence, provided that the amino acid substitutions are maintained and the protein has immunoglobulin binding activity. [3] A protein comprising the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, wherein the protein is selected from (i) or (ii) below: (i) A protein having the amino acid substitutions (3) to (9) and (11) to (18) of the amino acid sequence described in SEQ ID NO: 1, and in which the 38th lysine residue of SEQ ID NO: 1 is maintained. (ii) A protein having immunoglobulin-binding activity, comprising an amino acid sequence that has 70% or more identity with the entire amino acid sequence including the amino acid substitutions (3) to (9) and (11) to (18) described in Sequence ID No. 1, provided that the amino acid substitutions and the 38th lysine residue of Sequence ID No. 1 are maintained. [4] A polynucleotide comprising a base sequence encoding the protein described in [1] to [3] above. [5] A vector comprising the polynucleotide described in [4] above. [6] A transformer comprising the vector described in [5] above. [7] The transformant according to [6], wherein the host of the transformant is Escherichia coli. [8] A step of culturing the transformant described in [5] or [6] above and expressing a protein, A method for producing a protein, comprising the step of recovering the aforementioned protein. [9] An immunoglobulin adsorbent comprising an insoluble carrier and the proteins described in [1] to [3] above immobilized on the insoluble carrier.

[10] A column packed with the adsorbent described in [9] above.

[11] A step of adding a solution containing an antibody to the column described in

[10] above, and adsorbing the antibody onto the adsorbent, A method for separating an antibody, comprising the step of eluting the antibody adsorbed onto the adsorbent. [Effects of the Invention]

[0010] According to the present disclosure, a protein having immunoglobulin-binding activity that can be used as a ligand protein of an immunoglobulin adsorbent which enables elution of antibodies under milder pH conditions than conventional immunoglobulin adsorbents can be provided. Mode for Carrying Out the Invention

[0011] Hereinafter, an example of an embodiment of the protein of the present disclosure will be described and explained.

[0012] In the present embodiment, in accordance with common practice, the amino acid sequences of peptides are described such that the amino terminus (hereinafter also referred to as the "N-terminus") is located on the left side and the carboxyl terminus (hereinafter also referred to as the "C-terminus") is located on the right side.

[0013] In the present embodiment, the positions "before" and "after" a specific position in an amino acid sequence refer to the positions adjacent to the N-terminal side and the C-terminal side of the specific position, respectively. For example, when inserting an amino acid residue into a position "before" or "after" a specific position, the inserted amino acid residue is arranged at a position adjacent to the N-terminal side or C-terminal side of the specific position, respectively.

[0014] In the present embodiment, the term "amino acid sequence N" refers to the amino acid sequence set forth in SEQ ID NO: N, where N is an integer. For example, in the case of the amino acid sequence set forth in SEQ ID NO: 1, it may be referred to as amino acid sequence 1.

[0015] In the present embodiment, the term "base sequence n" refers to the base sequence set forth in SEQ ID NO: n, where n is an integer. For example, in the case of the base sequence set forth in SEQ ID NO: 2, it may be referred to as base sequence 2.

[0016] The protein of this embodiment is a protein containing the amino acid sequence of the immunoglobulin-binding domain of Protein L (hereinafter also referred to as "FpL") derived from bacteria of the genus Finegoldia, wherein the amino acid sequence has one or more amino acid substitutions selected from T25H (this notation indicates that the amino acid residue corresponding to the 25th threonine in amino acid sequence 1 is substituted with histidine; other amino acid substitutions shall be interpreted similarly hereinafter) and K29H. Because the amino acid sequence of the immunoglobulin-binding domain of FpL has one or more amino acid substitutions selected from T25H and K29H, it can be used as a ligand protein for an immunoglobulin adsorbent that allows antibody elution under milder pH conditions than conventional immunoglobulin adsorbents. That is, the protein of this embodiment is an immunoglobulin-binding protein that can be used as a ligand protein for an immunoglobulin adsorbent that allows antibody elution under milder pH conditions compared to an immunoglobulin adsorbent containing an immunoglobulin-binding protein that does not have one or more amino acid substitutions selected from T25H and K29H.

[0017] Examples of the genus Finegoldia include Finegoldia magna, and the protein of this embodiment is preferably derived from Finegoldia magna.

[0018] In this embodiment, "immunoglobulin-binding domain" refers to a functional unit of a polypeptide contained in an immunoglobulin-binding protein that independently possesses immunoglobulin-binding activity. The immunoglobulin-binding activity can be determined by a contact test between the protein and a carrier containing immunoglobulin. In this contact test, for example, the protein is brought into contact with a carrier containing immunoglobulin, the unreacted fraction is removed, and then the protein is eluted from the carrier. The absorbance of the obtained eluate at a wavelength of 280 nm is measured to confirm whether the protein is present, that is, whether the protein possesses immunoglobulin-binding activity.

[0019] Examples of immunoglobulin-binding domains of Protein L derived from Finegoldia magna include domain B1, domain B2, domain B3, domain B4, domain B5, domain C1, domain C2, domain C3, domain C4, and their variants. Of these, domains B5, C3, and C4 are preferred, with domain C3 being more preferred.

[0020] Examples of Finegoldia magna-derived Protein L include: Domain B1: amino acid residues from positions 104 to 173 of amino acid sequence 17; Domain B2: amino acid residues from positions 176 to 245 of amino acid sequence 17; Domain B3: amino acid residues from positions 248 to 317 of amino acid sequence 17; Domain B4: amino acid residues from positions 320 to 389 of amino acid sequence 17; Domain B5: amino acid residues from positions 393 to 462 of amino acid sequence 17; Domain C1: amino acid residues from positions 249 to 317 of amino acid sequence 18; Domain C2: amino acid residues from positions 320 to 389 of amino acid sequence 18; Domain C3: amino acid residues from positions 394 to 463 of amino acid sequence 18 (amino acid sequence 1); and Domain C4: amino acid residues from positions 468 to 537 of amino acid sequence 18.

[0021] The protein of this embodiment is preferably a protein selected from (a) or (b) below; (a) Proteins containing amino acid substitutions of T25H, E4G, P6S, K7A, K13R, K22R, I23R, K29I, K38E, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R and A69V in amino acid sequence 1 (hereinafter also referred to as "amino acid substitution 10c"), or amino acid substitutions of K29H, E4G, P6S, K7A, K13R, K22R, I23R, K38E, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R and A69V (hereinafter also referred to as "amino acid substitution 10d"), (b) A protein comprising an amino acid sequence of amino acid sequence 1 that has 70% or more identity with the entire amino acid sequence including amino acid substitution 10c or amino acid substitution 10d, wherein the amino acid substitution is maintained and the protein has immunoglobulin binding activity.

[0022] As described in (a) above, the protein of this embodiment is preferably a protein containing amino acid substitution 10c (hereinafter also referred to as "protein 10c of this embodiment") or a protein containing amino acid substitution 10d (hereinafter also referred to as "protein 10d of this embodiment") in amino acid sequence 1. The protein 10c of this embodiment is preferably a protein containing amino acid sequence 7. Furthermore, the protein 10d of this embodiment is preferably a protein containing amino acid sequence 9.

[0023] In this embodiment, protein 10c contains an amino acid substitution 10c in the immunoglobulin-binding domain of FpL, allowing the antibody to be eluted under milder pH conditions. Examples of protein 10c in this embodiment include a polypeptide consisting of one amino acid sequence 7, or a polypeptide in which two or more amino acid sequences 7 are linked in a linear chain. The number of amino acid sequences 7 contained in protein 10c in this embodiment is preferably 1 to 10, more preferably 1 to 5.

[0024] The protein 10d of this embodiment allows for antibody elution under milder pH conditions because the immunoglobulin-binding domain of FpL contains amino acid substitution 10d. The protein 10d of this embodiment may be, for example, a polypeptide consisting of one amino acid sequence 9, or a polypeptide in which two or more amino acid sequences 9 are linked in a linear chain. The number of amino acid sequences 9 contained in the protein 10d of this embodiment may be preferably 1 to 10, more preferably 1 to 5.

[0025] In this embodiment, "linearly linked" may refer to a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are linked in series by a peptide bond.

[0026] As described in (b) above, it is preferable that the protein of this embodiment includes an amino acid sequence having 70% or more identity with respect to the entire amino acid sequence including amino acid substitution 10c, provided that amino acid substitution 10c is maintained and the protein has immunoglobulin binding activity (hereinafter also referred to as "protein 10c variant of this embodiment"), or includes an amino acid sequence having 70% or more identity with respect to the entire amino acid sequence including amino acid substitution 10d, provided that amino acid substitution 10d is maintained and the protein has immunoglobulin binding activity (hereinafter also referred to as "protein 10d variant of this embodiment").

[0027] The protein 10c variant and the protein 10d variant of this embodiment may have an identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more, in the immunoglobulin-binding domain of FpL with respect to the entire amino acid sequence.

[0028] In this embodiment, "identity to the entire amino acid sequence" refers to the ratio of amino acid residues of the same type to the entire amino acid sequence. The identity of the amino acid sequence can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0029] Another embodiment of the protein of this embodiment is a protein comprising the amino acid sequence of the immunoglobulin-binding domain of FpL, wherein the protein is selected from (i) or (ii) below; (i) A protein containing the amino acid substitutions E4G, P6S, K7A, K13R, K22R, I23R, K29I, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R, and A69V in amino acid sequence 1 (hereinafter also referred to as "amino acid substitution 9f"), in which the 38th lysine residue of amino acid sequence 1 is maintained. (ii) A protein that contains an amino acid sequence of amino acid sequence 1 that has 70% or more identity with the entire amino acid sequence including amino acid substitution 9f, provided that amino acid substitution 9f and the 38th lysine residue of sequence number 1 are maintained, and that has immunoglobulin binding activity.

[0030] The protein of this embodiment contains the amino acid substitution 9f and maintains the lysine residue at position 38 of amino acid sequence 1, making it usable as a ligand protein for immunoglobulin adsorbents that allows antibody elution under milder pH conditions than conventional immunoglobulin adsorbents.

[0031] As described in (i) above, it is preferable that the protein of this embodiment is a protein in which amino acid substitution 9f is included in amino acid sequence 1 and the 38th lysine residue of amino acid sequence 1 is maintained (hereinafter also referred to as "protein 9f of this embodiment"). It is preferable that protein 9f of this embodiment is a protein that includes amino acid sequence 11.

[0032] In this embodiment, protein 9f contains an amino acid substitution 9f in the immunoglobulin-binding domain of FpL, maintaining the 38th lysine residue of amino acid sequence 1, thereby enabling antibody elution under milder pH conditions. Examples of protein 9f in this embodiment include a polypeptide consisting of amino acid sequence 11, or a polypeptide in which two or more amino acid sequences 11 are linked in a linear chain. The number of amino acid sequences 11 contained in protein 9f in this embodiment is preferably 1 to 10, more preferably 1 to 5.

[0033] As described in (ii) above, the protein of this embodiment preferably contains an amino acid sequence that has an identity of 70% or more with respect to the entire amino acid sequence including amino acid substitution 9f, provided that amino acid substitution 9f and the 38th lysine residue of amino acid sequence 1 are maintained, and the protein has immunoglobulin-binding activity (hereinafter also referred to as the "protein 9f variant of this embodiment").

[0034] In this embodiment, the protein 9f variant has sufficient, preferably 70% or more, 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more, in the immunoglobulin-binding domain of FpL to have identity with respect to the entire amino acid sequence.

[0035] The protein of this embodiment can be immobilized on an insoluble carrier and used as an adsorbent (hereinafter also referred to as "the adsorbent of this embodiment") that adsorbs immunoglobulins (hereinafter also referred to as "antibodies"), and can also be used in a method for separating antibodies using the adsorbent of this embodiment.

[0036] The adsorbent of this embodiment may be an insoluble carrier on which a protein containing the amino acid sequence of the immunoglobulin-binding domain of FpL has been immobilized, which is a protein having one or more amino acid substitutions selected from T25H and K29H, or a protein containing amino acid substitution 9f and maintaining the 38th lysine residue of amino acid sequence 1. Compared to an insoluble carrier on which the immunoglobulin-binding domain of Protein L without the aforementioned amino acid substitutions is immobilized, the adsorbent of this embodiment can elute antibodies under mild pH conditions.

[0037] There are no particular limitations on the method of separating antibodies using the adsorbent of this embodiment, but a chromatography method using a column obtained by packing an empty column with the adsorbent of this embodiment is preferred. In this embodiment, antibody separation is not limited to the separation of antibodies in the presence of contaminants, but may also include the separation of antibodies based on their structure, properties, or activity.

[0038] As an example of the chromatography separation method, an antibody is separated by adding a solution containing an antibody to a column packed with the adsorbent of this embodiment, adsorbing the antibody onto the adsorbent, and then eluting the antibody adsorbed onto the adsorbent. That is, this embodiment discloses a method for separating antibodies contained in an eluate, comprising the steps of adding a solution containing an antibody to a column packed with the adsorbent of this embodiment to adsorb the antibody onto the adsorbent (hereinafter also referred to as the "adsorption step") and eluting the antibody adsorbed onto the adsorbent (hereinafter also referred to as the "elution step").

[0039] For example, a pump can be used to deliver the antibody-containing solution. The antibody-containing solution may be pre-vaporized using an appropriate buffer before being added to the column. Alternatively, the column may be equilibrated using an appropriate buffer before adding the antibody-containing solution. Equilibration of the column allows for the separation of antibodies with higher purity. Examples of buffers used for solvent replacement and equilibration include one or more selected from the group consisting of phosphate buffer, acetate buffer, and MES buffer. A salt of 1 mmol / L to 1000 mmol / L may be added to the buffer. Examples of salts include sodium chloride, sodium acetate, or magnesium chloride. The buffer used for solvent replacement may be the same as, or different from, the buffer used for equilibration.

[0040] If contaminants remain on the column after passing an antibody-containing solution through it, it is preferable to remove the contaminants from the column before eluting the antibodies adsorbed on the antibody adsorbent. Contaminants can be removed from the column, for example, using an appropriate buffer solution. The buffer solution used for removing contaminants can be similarly described to the buffer solutions used for solvent replacement and equilibration.

[0041] Antibodies adsorbed to the adsorbent of this embodiment can be eluted by weakening the interaction between the antibody and the protein of this embodiment. Examples of methods for weakening the interaction include changing the pH, adding a counterpeptide, increasing the temperature, and changing the salt concentration, with pH changes being preferred. An example of a pH change is a decrease in pH. An example of a decrease in pH is a decrease in pH using a buffer. Antibodies adsorbed to the adsorbent of this embodiment can be eluted using an eluate. An example of an eluate is a buffer that is more acidic than the buffer used for solvent exchange or equilibration. Examples of acidic buffers include citrate buffer, glycine hydrochloride buffer, or acetate buffer. That is, the pH can be lowered by using the acidic buffer. The pH of the eluate can be set within a range that does not impair the function of the antibody. The pH of the eluate may be, for example, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 2.95 or higher, 3 or higher, 3.05 or higher, 3.1 or higher, or 3.2 or higher, and may also be 4 or lower, 3.5 or lower, 3.3 or lower, 3.2 or lower, or 3.1 or lower. Any combination of these upper and lower limits is acceptable. Therefore, the pH of the eluate may be, for example, 2.5 or higher and 4 or lower, 2.8 or higher and 4 or lower, 2.95 or higher and 4 or lower, 3 or higher and 4 or lower, or 3.05 or higher and 4 or lower.

[0042] In this embodiment, "mild pH conditions" means pH conditions close to neutral. That is, "antibody elution is possible under mild pH conditions" means that antibody elution is possible under pH conditions close to neutral. Also, since the pH of a typical eluate can be acidic, "antibody elution is possible under mild pH conditions" means that the pH is increasing at the time of antibody elution. In this embodiment, the time of antibody elution means the time when a perpendicular line is drawn from the peak top in the chromatogram. When eluting antibodies adsorbed in the adsorption step, antibodies can usually be eluted at a pH lower than the pH in the adsorption step, so antibodies may be eluted by a pH gradient, and the earlier the time of antibody elution compared to conventional methods, the milder the pH conditions under which the antibodies can be eluted. Methods for lowering the pH of the eluate include changing the pH in two or more stages (hereinafter also called a "step gradient"), or changing it with a linear slope (hereinafter also called a "linear gradient"), and a linear gradient is preferred.

[0043] The insoluble carrier is preferably a carrier made from a polysaccharide or a synthetic polymer. Examples of the polysaccharide include agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, or starch. Examples of the synthetic polymer include polyvinyl alcohol, polymethacrylate, polyacrylamide, polystyrene, or polyurethane. An example of the insoluble carrier is a polymethacrylate gel, agarose gel, or cellulose gel into which hydroxyl groups have been introduced.

[0044] The insoluble carrier can take the form of particulate matter, film, or fibers, with particulate matter being preferred. If the insoluble carrier is particulate matter, the particle size is preferably 20 μm or more and 300 μm or less. The insoluble carrier may be porous or non-porous.

[0045] One method for immobilizing the adsorbent of this embodiment onto an insoluble carrier is to bring the protein of this embodiment into contact with the insoluble carrier. This allows the amino groups of the protein and the active groups of the insoluble carrier to form a covalent bond. Examples of active groups on the insoluble carrier include N-hydroxysuccinimide (NHS) activated ester groups, epoxy groups, carboxyl groups, maleimide groups, haloacetyl groups, tresyl groups, formyl groups, or iodine groups, with formyl groups being preferred. The insoluble carrier having active groups may be a commercially available carrier as is, or a carrier in which the desired active groups have been introduced onto the surface of the insoluble carrier may be used.

[0046] Examples of compounds that introduce a desired active group to the surface of the insoluble carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic anhydride, bromoacetic anhydride, iodoacetic acid anhydride, 2-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, or 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide, glutaraldehyde, sodium periodate, and ruthenium tetroxide, which react with hydroxyl groups or amino groups on the surface of the insoluble carrier.

[0047] In this embodiment, the protein and the insoluble carrier can be brought into contact by dissolving the protein in a buffer solution and then bringing it into contact with the active group of the insoluble carrier. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, Tris buffer, or borate buffer.

[0048] The reaction temperature when immobilizing the protein of this embodiment onto an insoluble support can be appropriately set according to the reactivity of the active group and the stability of the protein of the present invention, preferably 5°C to 50°C, and more preferably 10°C to 35°C.

[0049] The antibody adsorbed to the adsorbent of this embodiment is not particularly limited as long as it is a protein that can bind to the protein of this embodiment, but it is preferably a protein having a κ light chain. Examples of antibodies include IgG, IgM, IgA, IgD, or IgE. If the antibody is IgG, it may be any of IgG1, IgG2, IgG3, or IgG4. The antibody may also be either a monoclonal antibody or a polyclonal antibody. The antibody may be derived from a single organism or from a combination of two or more organisms. Furthermore, examples of antibodies include antibodies with artificially modified structures, such as bispecific antibodies, fusion antibodies of a κ light chain and another protein, or complexes of a κ light chain and a drug (ADC).

[0050] Next, the method for producing the protein according to this embodiment will be described.

[0051] The protein of this embodiment is obtained by a manufacturing method comprising the steps of culturing a transformant containing a polynucleotide encoding the protein of this embodiment and expressing the protein, and recovering the protein.

[0052] (Protein expression process) The method for producing the protein of this embodiment includes a step of culturing a transformant containing a polynucleotide encoding the protein of this embodiment and expressing the protein (hereinafter also referred to as the "protein expression step").

[0053] A transformant containing the polynucleotide encoding the protein of this embodiment (hereinafter also referred to as "the oligonucleotide of this embodiment") can be obtained by a known manufacturing method, and an example of such a method is one that includes the step of transforming a host with a vector containing the polynucleotide of this embodiment to obtain a transformant.

[0054] The polynucleotide-containing vector in this embodiment is not particularly limited as long as it can be stably present and replicated within the transforming host. Examples include pET plasmid vectors, pUC plasmid vectors, pTrc plasmid vectors, pCDF plasmid vectors, or pBBR plasmid vectors, with pET plasmid vectors being preferred.

[0055] When inserting the polynucleotide of this embodiment into the vector, it is preferable to insert it in a state in which it is linked to a functional polynucleotide necessary for the expression of the protein encoded by the polynucleotide of this embodiment. Examples of such functional polynucleotides include one or more selected from the group consisting of trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, temperature-shift promoter, and IPTG-inducible promoter. If the vector contains an IPTG-inducible promoter, an example of an inducer is IPTG (Isopropyl β-D-1-thiogalactopyranoside). Various conditions for IPTG induction can be carried out under conditions well known in the art.

[0056] The method for transforming a host with a vector containing the polynucleotides of this embodiment (i.e., a vector containing the polynucleotides of this embodiment) can be carried out using methods commonly used by those skilled in the art. The transformation method can be selected according to the cells to be introduced, and examples include heat shock (chemical transformation) or electroporation of chemically prepared competent cells.

[0057] The polynucleotide-containing vector of this embodiment preferably contains base sequence 8, base sequence 10, or base sequence 12.

[0058] Examples of the host include bacteria, yeast, insect cells, animal cells, or plant cells. However, bacteria are preferred, and Escherichia coli is more preferred, due to their ease of handling, ease of cultivation, feasibility of high-density culture, and the availability of hosts and vectors for genetic manipulation. Examples of Escherichia coli strains used for transformation include JM109, JM110, BL21, or BL21(DE3), with BL21(DE3) being preferred.

[0059] In the protein expression process, the culture of the transformants may be carried out according to known methods, and examples include individual culture or liquid culture. Examples of liquid culture include one or more selected from the group of shaking culture, jar fermenter culture, and tank culture, with shaking culture being preferred. Furthermore, the culture of the transformants may be carried out under at least one of anaerobic or aerobic conditions, with aerobic conditions being preferred. The culture temperature can be appropriately selected from the range in which the host grows, and is preferably between 15°C and 40°C.

[0060] In this embodiment, the transformants can be cultured in a medium suitable for culturing the host organism, and LB (Luria-Bertani) medium, TB (Terrific broth) medium, or 2×YT medium supplemented with the necessary nutrients are preferred. Furthermore, in order to selectively grow transformants depending on whether or not the polynucleotide of this embodiment is introduced into the vector, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the culture medium. For example, if the vector contains a kanamycin resistance gene, kanamycin should be added to the culture medium.

[0061] (Protein recovery process) The protein production method of this embodiment includes a step of recovering the protein obtained in the protein expression step (hereinafter also referred to as the "protein recovery step").

[0062] To recover the protein of this embodiment from the culture medium containing the transformants, an appropriate recovery method can be selected depending on the mode of protein expression. If the protein is expressed in the culture supernatant of the culture medium, the microorganisms can be separated by centrifugation, and a solution containing the protein of this embodiment can be recovered from the resulting culture supernatant. On the other hand, if the protein is expressed intracellularly, the cells can be collected by centrifugation, then the cells can be lysed by adding an enzyme treatment agent or surfactant, and the resulting lysate can be recovered by removing impurities by centrifugation or membrane filtration to obtain the protein of this embodiment.

[0063] To recover a higher purity protein, further separation and purification methods using liquid chromatography may be performed. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, or affinity chromatography. By fractionating using such chromatography, the protein of this embodiment can be prepared to a high purity. [Examples]

[0064] The present disclosure will be described below with reference to examples. However, the present disclosure is not limited to these examples.

[0065] Comparative Example 1 Using the method disclosed in Japanese Patent Publication No. 2023-103953 as a reference, a vector pET-FpL_C3KX 9a was constructed encoding a protein (hereinafter also referred to as "FpL_C3KX 9a") in which amino acid substitutions of E4G, P6S, K7A, K13R, K22R, I23R, K29I, K38E, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R, and A69V were introduced into the amino acid residue corresponding to the immunoglobulin-binding domain C3 (amino acid sequence 1) of Protein L (GenBank No. AAA67503) derived from natural Finegoldia magna. The amino acid sequence of FpL_C3KX 9a is shown in Sequence ID No. 3, and the nucleotide sequence encoding FpL_C3KX 9a is shown in Sequence ID No. 4.

[0066] Furthermore, referring to the method disclosed in Japanese Patent Publication No. 2023-103953, a vector pET-FpL_C3KX 10a was prepared that encodes a protein (hereinafter also referred to as "FpL_C3KX 10a") in which the amino acid substitutions E4G, P6S, K7A, K13R, K22R, I23R, K29I, K38E, N44R, K48H, E49D, N50Y, Y53F, N62Y, I64L, K67R, and A69V were introduced into amino acid sequence 1. The amino acid sequence of FpL_C3KX 10a is shown in Sequence ID No. 5, and the nucleotide sequence encoding FpL_C3KX 10a is shown in Sequence ID No. 6.

[0067] In other words, the following was performed in this comparative example.

[0068] (1) A polynucleotide containing base sequence 4 encoding amino acid sequence 3 and a polynucleotide containing base sequence 6 encoding amino acid sequence 5 were synthesized. During synthesis, the recognition sequence for restriction enzyme NcoI (5'-CCATGG-3') was added to the 5' end, and the nucleotide sequence encoding six histidine residues (5'-CATCACCACCATCACCAC-3'), a stop codon, and the recognition sequence for restriction enzyme HindIII (5'-AAGCTT-3') were added to the 3' end.

[0069] (2) The synthesized polynucleotides containing base sequence 4 or base sequence 6 were treated with restriction enzymes NcoI and HindIII, and the size band of the target product was confirmed by agarose gel electrophoresis. After excising the target band, the polynucleotides were purified using the QIAquick Gel Extraction kit (QIAGEN). The purified polynucleotides were ligated with plasmid pET-26b, which had been previously digested with restriction enzymes NcoI and HindIII, using the DNA Ligation Kit (Takara Bio Inc.) to obtain ligation products. Escherichia coli BL21 (DE3) strain was transformed using these ligation products and cultured in LB plate medium containing 50 μg / mL kanamycin at 37°C for 16 hours to obtain transformants (genetically modified Escherichia coli).

[0070] (3) The transformants obtained in (2) were cultured in LB medium containing 50 μg / mL kanamycin, and then purified using the QIAprep Spin Miniprep kit (QIAGEN) to obtain a vector capable of expressing FpL_C3KX 9a or FpL_C3KX 10a.

[0071] Example 1 Using pET-FpL_C3KX 9a as a template, vectors containing the following polynucleotides (A), (B), or (C) were constructed by introducing nucleotide substitutions to the polynucleotide encoding FpL_C3KX 9a (base sequence 4) via inverse PCR. The mutations in amino acid sequence 1 are shown in Table 1. (A) A polynucleotide (base sequence 8) encoding FpL_C3KX 10c (amino acid sequence 7). (B) A polynucleotide (base sequence 10) encoding FpL_C3KX 10d (amino acid sequence 9). (C)FpL_C3KX A polynucleotide (base sequence 12) encoding 9f (amino acid sequence 11).

[0072] Reference example 1 Using pET-FpL_C3KX 9a as a template, vectors containing the following polynucleotides (D) or (E) were constructed by introducing nucleotide substitutions to the polynucleotide encoding FpL_C3KX 9a (SEQ ID NO: 4) via inverse PCR. The mutations relative to SEQ ID NO: 1 are shown in Table 1. (D) A polynucleotide (sequence number 14) encoding FpL_C3KX 10e (sequence number 13). (E)FpL_C3KX 10f (SEQ ID NO: 15) is a polynucleotide (SEQ ID NO: 16) that codes for this sequence.

[0073] [Table 1]

[0074] Example 2: Preparation of amino acid-substituted immunoglobulin-binding proteins (1) Transformants were prepared by transforming Escherichia coli BL21(DE3) strain (manufactured by Nippon Gene Co., Ltd.) using vectors encoding proteins into which amino acid substitutions were introduced, which were prepared in Comparative Example 1, Example 1, and Reference Example 1.

[0075] (2) The transformants prepared in (1) were inoculated into 2 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin, respectively, and pre-cultured by aerobic shaking at 37°C overnight to obtain pre-culture solutions.

[0076] (3) 200 μL of the pre-culture solution from (2) was inoculated into 20 mL of 2×YT liquid medium to which 50 μg / mL of kanamycin had been added, and the culture was incubated aerobically with shaking at 37°C to obtain the culture medium.

[0077] (4) Two hours after the start of culture, the culture temperature was changed to 20°C, and IPTG (IsoPropyl β-D-1-ThioGalactopyranoside) was added to a final concentration of 0.01 mmol / L. The culture was then continued at 20°C overnight under aerobic shaking conditions to obtain the culture medium.

[0078] (5) After the culturing was complete, bacterial cells were obtained from the culture medium of (4) by centrifugation. A protein extract was prepared from the obtained bacterial cells using BugBuster Protein Extraction Reagent (Merck). The extract was centrifuged, and the resulting supernatant was passed through a filter to obtain a clarified protein extract.

[0079] (6) The protein extract clarified in (5) was added to a column packed with Ni-NTA agarose (manufactured by Fujifilm Corporation) that had been pre-equilibrated with 50 mmol / L Tris buffer (pH 7.5) containing 500 mmol / L NaCl and 20 mmol / L imidazole (hereinafter also referred to as "wash buffer"). Subsequently, the column was washed with 10 times the volume of wash buffer, and the fraction corresponding to immunoglobulin-binding proteins (hereinafter also referred to as "immunoglobulin-binding protein solution") was recovered by passing 50 mmol / L Tris buffer (pH 7.5) containing 500 mmol / L NaCl and 500 mmol / L imidazole (hereinafter also referred to as "elution buffer").

[0080] (6) The absorbance of the recovered immunoglobulin-binding protein solution at a wavelength of 280 nm was measured to quantify the immunoglobulin-binding protein contained in the fraction. The immunoglobulin-binding protein solution was then prepared with elution buffer to a concentration of 0.5 mg / mL. Example 3: Preparation of an antibody isolation column (1) A polymethacrylate gel (Toyoparl, manufactured by Tosoh Corporation) having formyl groups as active groups on its surface was used as an insoluble carrier. The slurry of the carrier was filtered by suction on a glass filter and then dried by suction to prepare a suction-dried gel.

[0081] (2) Using the suction dry gel prepared in (1), the water content and bulk density were calculated using the following method. First, the water content per 1 g of suction dry gel was measured using a heat drying moisture meter (ML50, A&D Corporation), and the dry gel weight (g) after removing the water from the suction dry gel was calculated based on this. Then, 1 g of suction dry gel was prepared as an aqueous slurry, and the volume of the settled gel (mL) was measured by transferring it to a graduated cylinder and letting it stand. The bulk density (g / mL) was calculated by dividing the previously calculated dry gel weight by the settled gel.

[0082] (3) Based on the calculation results in (2), the suction dry gel prepared in (1) was measured into an Erlenmeyer flask so that the volume of the insoluble carrier was 2 mL. Appropriate amounts of a 100 mg / mL human-derived polyclonal antibody solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a 200 mmol / L borate buffer, and a 4 mol / L sodium chloride aqueous solution were added to the flask, and the mixture was shaken at 25°C for 4 hours to bind the insoluble carrier and the polyclonal antibody by Schiff base formation.

[0083] (4) After the reaction in (3) was complete, an appropriate amount of 1.1 mol / L aqueous dimethylamine borane solution was added and the remaining Schiff base was reduced by shaking at 25°C for 2 hours.

[0084] After the reaction in (5)(4) was completed, an appropriate amount of 3.6 mol / L 2-aminoethanol aqueous solution was added to block the formyl groups remaining on the insoluble carrier, thereby obtaining an antibody adsorbent.

[0085] (6) The obtained gel was packed into a stainless steel column with a diameter of 4.6 mm and a length of 35 mm, and an antibody separation column was prepared by immobilizing polyclonal antibodies on it. Example 4: Separation of antibodies by pH gradient The following buffer solutions were used for antibody isolation.

[0086] Buffer A: 100 mmol / L phosphate buffer (pH 7.0) Buffer B: 100 mmol / L citrate buffer (pH 2.2) (1) After equilibrating the antibody separation column prepared in Example 3 with buffer A, 100 μL of each immunoglobulin-binding protein solution prepared in Example 2(6) at a concentration of 0.5 mg / min was delivered. The flow rate was 0.3 mL / min.

[0087] (2) After delivering five column volumes of buffer A, the immunoglobulin-binding proteins adsorbed to the antibody separation column were eluted by linear gradient elution, where buffer B increased from 0% to 100% over 25 minutes. The eluted immunoglobulin-binding proteins were detected by measuring the absorbance at a wavelength of 280 nm. The obtained chromatograms were analyzed, and the elution position with the highest absorbance was defined as the retention time. The retention time was set to 0 minutes when five column volumes of buffer A had been delivered.

[0088] The results are shown in Table 2. Peaks were observed earlier for FpL_C3KX 10c, FpL_C3KX 10d, and FpL_C3KX 9f than for FpL_C3KX 9a and FpL_C3KX 10a. In other words, FpL_C3KX 10c, FpL_C3KX 10d, and FpL_C3KX 9f dissociated from antibodies at a higher pH than FpL_C3KX 9a and FpL_C3KX 10a. Therefore, immunoglobulin adsorbents using FpL_C3KX 10c, FpL_C3KX 10d, or FpL_C3KX 9f as ligand proteins can elute antibodies under milder pH conditions than immunoglobulin adsorbents using FpL_C3KX 9a or FpL_C3KX 10a as ligand proteins. From this, it was found that immunoglobulin-binding proteins having at least one amino acid substitution selected from at least T25H and K29H in amino acid sequence 1 elute antibodies under milder pH conditions compared to FpL_C3KX 9a and FpL_C3KX 10a. Furthermore, it was found that immunoglobulin-binding proteins having the amino acid substitutions E4G, P6S, K7A, K13R, K22R, I23R, K29I, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R, and A69V in amino acid sequence 1, and maintaining the 38th lysine residue of SEQ ID NO: 1, elute antibodies under milder pH conditions compared to FpL_C3KX 9a and FpL_C3KX 10a.

[0089] [Table 2]

Claims

1. A protein comprising the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, wherein the amino acid sequence has one or more amino acid substitutions selected from (1) and (2) below: (1) The amino acid residue corresponding to the 25th threonine in Sequence ID No. 1 is replaced with histidine. (2) The amino acid residue corresponding to the 29th lysine residue in SEQ ID NO: 1 is replaced with histidine.

2. The protein according to claim 1, selected from (a) or (b) below: (a) A protein comprising the amino acid sequence described in Sequence ID No. 1, including the amino acid substitutions in (1) and (3) to (18) below, or the amino acid substitutions in (2) and (3) to (8) and (10) to (18) below; (3) The amino acid residue corresponding to the fourth glutamic acid in SEQ ID NO: 1 is replaced with glycine. (4) The amino acid residue corresponding to the sixth proline in SEQ ID NO: 1 is replaced with serine. (5) The amino acid residue corresponding to the seventh lysine residue in Sequence ID No. 1 is replaced with alanine. (6) The amino acid residue corresponding to the 13th lysine residue in SEQ ID NO: 1 is replaced with arginine. (7) The amino acid residue corresponding to the 22nd lysine residue in Sequence ID No. 1 is replaced with arginine. (8) The amino acid residue corresponding to isoleucine at position 23 of Sequence ID No. 1 is replaced with arginine. (9) The amino acid residue corresponding to the 29th lysine residue in Sequence ID No. 1 is replaced with isoleucine. (10) The amino acid residue corresponding to the 38th lysine residue of Sequence ID No. 1 is replaced with glutamic acid. (11) The amino acid residue corresponding to asparagine at position 44 of Sequence ID No. 1 is replaced with arginine. (12) The amino acid residue corresponding to the 48th lysine residue of Sequence ID No. 1 is replaced with arginine. (13) The amino acid residue corresponding to glutamic acid at position 49 of SEQ ID NO: 1 is replaced with aspartic acid. (14) The amino acid residue corresponding to asparagine at position 50 of SEQ ID NO: 1 is replaced with tyrosine. (15) The amino acid residue corresponding to tyrosine at position 53 of Sequence ID No. 1 is substituted with phenylalanine. (16) The amino acid residue corresponding to asparagine at position 62 of SEQ ID NO: 1 is replaced with tyrosine. (17) The amino acid residue corresponding to lysine at position 67 of Sequence ID No. 1 is replaced with arginine. (18) The amino acid residue corresponding to alanine at position 69 of SEQ ID NO: 1 is substituted with valine. (b) A protein comprising an amino acid sequence described in Sequence ID No. 1, wherein the amino acid substitutions of (1) and (3) to (18), or the amino acid substitutions of (2) to (8) and (10) to (18), are identical to the entire amino acid sequence, provided that the amino acid substitutions are maintained and the protein has immunoglobulin binding activity.

3. A protein comprising the amino acid sequence of the immunoglobulin-binding domain of Protein L derived from a bacterium of the genus Finegoldia, wherein the protein is selected from (i) or (ii) below: (i) A protein having the amino acid substitutions (3) to (9) and (11) to (18) of the amino acid sequence described in SEQ ID NO: 1, and in which the 38th lysine residue of SEQ ID NO: 1 is maintained. (ii) A protein having immunoglobulin-binding activity, comprising an amino acid sequence that has 70% or more identity with the entire amino acid sequence including the amino acid substitutions (3) to (9) and (11) to (18) described in Sequence ID No. 1, provided that the amino acid substitutions and the 38th lysine residue of Sequence ID No. 1 are maintained.

4. A polynucleotide comprising a base sequence encoding the protein described in claim 1 or 3.

5. A vector comprising the polynucleotide described in claim 4.

6. A transformant comprising the vector described in claim 5.

7. The transformant according to claim 6, wherein the host of the transformant is Escherichia coli.

8. A step of culturing the transformant according to claim 6 and expressing a protein, A method for producing a protein, comprising the step of recovering the aforementioned protein.

9. An immunoglobulin adsorbent comprising an insoluble carrier and a protein according to claim 1 or 3 immobilized on the insoluble carrier.

10. A column packed with the adsorbent described in claim 9.

11. A step of adding a solution containing an antibody to the column according to claim 10, and adsorbing the antibody onto the adsorbent, A method for separating an antibody, comprising the step of eluting the antibody adsorbed onto the adsorbent.

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