Affinity carrier

Abstract

Provided are an immunoglobulin-binding protein containing a mutant immunoglobulin-binding domain and an affinity carrier containing the immunoglobulin-binding protein. The mutant immunoglobulin-binding domain comprises an amino acid sequence having at least 80% identity with any of the amino acid sequences represented by SEQ ID NOS: 1-3, and has the following mutations (a) and (b): (a) substitution of an amino acid residue at a position corresponding to position 3 in any of the amino acid sequences represented by SEQ ID NOS: 1-3 with Ala or Asp; and (b) insertion of at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to position 3 and the position corresponding to position 4 in any of the amino acid sequences represented by SEQ ID NOS: 1-3.

Description

Affinity carrier 【0001】 The present invention relates to an affinity carrier containing an immunoglobulin-binding protein. 【0002】 In the production process of biopharmaceuticals such as antibody drugs, it is necessary to separate a target pharmaceutical protein such as an antibody from an expression medium and purify it to a purity acceptable as a therapeutic or diagnostic agent. Reagent or pharmaceutical proteins are generally produced through purification by affinity chromatography. For affinity purification, a column immobilized with a ligand that specifically binds to immunoglobulin is used. As a ligand for affinity purification of antibodies, an immunoglobulin-binding protein derived from Protein A or a variant thereof is generally used. 【0003】 Patent Document 1 describes that an affinity carrier containing a mutant domain in which at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr is inserted between the 3rd and 4th positions with respect to the amino acid sequence of the B domain, Z domain, or C domain of Protein A as a ligand has improved alkali resistance and can maintain a high immunoglobulin-binding ability even when repeatedly used. Patent Document 2 describes that an affinity carrier containing a mutant domain in which Asn at the 3rd position is substituted with Gln, Ala, or Asp with respect to the C domain of Protein A as a ligand has improved alkali resistance and can maintain a high immunoglobulin-binding ability even when repeatedly used. 【0004】 International Publication No. 2016 / 152946 International Publication No. 2020 / 040307 【0005】Maintaining the yield and purity of the target substance even after repeated use is an important property for affinity carriers. To achieve this, it is important not only to improve the alkali resistance of the carrier but also to suppress the leakage of ligands from the carrier. Furthermore, since the proteins used as ligands for affinity carriers are generally produced by culture, it is important that these ligand proteins are not degraded in the culture medium. This is crucial for the efficiency of ligand production and, consequently, for the economic viability of the affinity carrier. 【0006】 The present invention provides an affinity carrier that is alkali-resistant, maintains high immunoglobulin binding ability even after repeated use, and suppresses ligand leakage. 【0007】The present invention provides the following as representative embodiments: (1) an affinity carrier comprising a solid carrier and an immunoglobulin-binding protein bound to the solid carrier, wherein the immunoglobulin-binding protein comprises at least one mutant immunoglobulin-binding domain, the mutant immunoglobulin-binding domain having an amino acid sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 1 to 3, and the mutant immunoglobulin-binding domain having the following mutations (a) and (b): (a) substitution of an amino acid residue with Ala or Asp at the position corresponding to the 3rd position of the amino acid sequence of any of SEQ ID NOs: 1 to 3; (b) An affinity carrier, which inserts at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to the 3rd and 4th positions of any of the amino acid sequences of SEQ ID NOs: 1 to 3.[2] The mutant immunoglobulin-binding domain further has at least one mutation selected from the group consisting of (c) to (j) below: (c) Substitution of an amino acid residue with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (d) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (e) Substitution of an amino acid residue with Ala or Asp at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (f) Substitution of an amino acid residue with Ala, Gln or Glu at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (g) Substitution of an amino acid residue with Ala at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (h) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (i) Substitution of an amino acid residue at the position corresponding to position 50 of any amino acid sequence of Sequence ID No. 1 to 3 with Ala or Arg; (j) Substitution of an amino acid residue at the position corresponding to position 58 of any amino acid sequence of Sequence ID No. 1 to 3 with Ala or Arg, the affinity carrier according to [1]. [3] The affinity carrier according to [2], wherein the mutant immunoglobulin binding domain has the mutations of (a), (b), (c), (e), and (g). [4] The affinity carrier according to [3], wherein the mutant immunoglobulin binding domain has the mutations of (a), (b), (c), (e), and (g), and at least one mutation selected from the group consisting of (d), (f), (h), (i), and (j). [5] The affinity carrier according to any one of [1] to [4], wherein the immunoglobulin binding protein contains 2 to 12 immunoglobulin binding domains.[6] An immunoglobulin-binding protein comprising at least one mutant immunoglobulin-binding domain, wherein the mutant immunoglobulin-binding domain consists of an amino acid sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 1 to 3, and the mutant immunoglobulin-binding domain has the following mutations: (a) substitution of an amino acid residue at the position corresponding to position 3 of the amino acid sequence of any of SEQ ID NOs: 1 to 3 with Ala or Asp; (b) insertion of at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to position 3 and position 4 of the amino acid sequence of any of SEQ ID NOs: 1 to 3, an immunoglobulin-binding protein. [7] The mutant immunoglobulin-binding domain further has at least one mutation selected from the group consisting of (c) to (j) below: (c) substitution of an amino acid residue with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (d) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (e) substitution of an amino acid residue with Ala or Asp at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (f) substitution of an amino acid residue with Ala, Gln or Glu at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (g) substitution of an amino acid residue with Ala at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (h) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (i) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (j) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; The immunoglobulin-binding protein described in [6].[8] The immunoglobulin-binding protein according to [7], wherein the mutant immunoglobulin-binding domain has the mutations of (a), (b), (c), (e), and (g). [9] The immunoglobulin-binding protein according to [8], wherein the mutant immunoglobulin-binding domain has the mutations of (a), (b), (c), (e), and (g), and at least one mutation selected from the group consisting of (d), (f), (h), (i), and (j).

[10] A polynucleotide encoding the immunoglobulin-binding protein according to any one of [6] to [9].

[11] A transformant into which the polynucleotide according to

[10] has been introduced.

[12] A method for isolating an antibody or a fragment thereof. A method comprising: (Step 1) passing a solution containing an antibody or a fragment thereof as a target substance through a chromatography carrier according to any one of (1) to (5) to bind the target substance to the carrier; (Step 2) recovering the target substance from the carrier; and (Step 3) passing an alkaline solution through the carrier.

[13] The method according to

[12] , comprising repeating steps 1 to 3 multiple times. 【0008】 The affinity carrier of the present invention exhibits excellent alkali resistance and suppresses ligand leakage, thereby maintaining high immunoglobulin binding ability even after repeated use, and preserving the purity of the purified target substance. Furthermore, the protein used as the ligand in the affinity carrier of the present invention is resistant to degradation in culture media, resulting in efficient manufacturing. Therefore, the affinity carrier of the present invention offers excellent cost performance. 【0009】 All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety. 【0010】 In this specification, notations representing numerical ranges such as "A to B" are synonymous with "greater than or equal to A, less than or equal to B," and include both A and B within that numerical range. 【0011】In this specification, the term "ligand" as used in relation to affinity chromatography refers to a molecule that binds to the target substance of affinity chromatography. "Protein ligand" refers to a ligand in which the portion that binds to the target substance is composed of a protein. 【0012】 In this specification, the identity of amino acid sequences and nucleotide sequences can be determined using the BLAST algorithm (Pro. Natl. Acad. Sci. USA., 1993, 90:5873-5877). Based on this BLAST algorithm, programs called BLASTN, BLASTX, BLASTP, TBLASTN, and TBLASTX have been developed (J. Mol. Biol., 1990, 215:403-410). When using these programs, the default parameters of each program can be used. The specific methods of these analysis methods are publicly known (see [www.ncbi.nlm.nih.gov]). 【0013】 In this specification, "at least 80%" with respect to the identity of amino acid sequences and nucleotide sequences means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. 【0014】In this specification, the "corresponding positions" on amino acid sequences and nucleotide sequences can be determined by aligning the target sequence and the reference sequence (for example, the amino acid sequence of SEQ ID NO: 1) to give maximum homology to the conserved amino acid residues or nucleotides present in each amino acid sequence or nucleotide sequence. Alignment can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Crystal W multiple alignment program (Thompson J.D et al., Nucleic Acids Res., 1994, 22:4673-4680) with default settings. Crystal W can be used, for example, on the website of the DNA Databank of Japan (DDBJ [www.ddbj.nig.ac.jp / index.html]) operated by the National Institute of Genetics. The position of the target sequence aligned to any position on the reference sequence by the above-described alignment is considered to be the "corresponding position" to that arbitrary position. 【0015】 In this specification, amino acid residues are also referred to by the following abbreviations: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). In this specification, the amino acid sequences of peptides are described in accordance with the conventional method, with the amino terminus (hereinafter referred to as the N terminus) on the left and the carboxyl terminus (hereinafter referred to as the C terminus) on the right. 【0016】In this specification, the “preceding” and “postceding” positions of a particular amino acid sequence refer to the positions adjacent to the N-terminus and C-terminus of that particular position, respectively. For example, when inserting an amino acid residue into the “preceding” and “postceding” positions of a particular position, the inserted amino acid residue is positioned at the positions adjacent to the N-terminus and C-terminus of that particular position, respectively. 【0017】 In this specification, "antibody" means a molecule that can specifically bind to a target such as a polypeptide via at least one antigen recognition site located in the variable region of an immunoglobulin. In this specification, "antibody" may include, for example, immunoglobulins of any class such as IgG, IgA, IgD, IgE, IgM, and their subclasses, fragments thereof containing an antigen recognition site (e.g., Fab, Fab', F(ab')2, Fv, rIgG, etc.), single-chain antibodies (ScFv), heavy-chain antibodies, and VHH antibodies. In this specification, "antibody" may also include chimeric antibodies such as humanized antibodies, antibody complexes, and other immunoglobulin modifications containing an antigen recognition site. In this specification, "antibody fragment" may be an antibody fragment containing an antigen recognition site or an antibody fragment that does not contain an antigen recognition site. Examples of antibody fragments that do not contain an antigen recognition site include proteins consisting only of the Fc region of an immunoglobulin, Fc fusion proteins, and their variants and modifications. 【0018】 In this specification, "immunoglobulin-binding domain" refers to a polypeptide chain contained in an immunoglobulin-binding protein that constitutes a functional unit having immunoglobulin (or antibody or antibody fragment) binding activity on its own. Preferred examples of the "immunoglobulin-binding domain" include the immunoglobulin-binding domain of protein A and its variants having immunoglobulin-binding activity. In this specification, "immunoglobulin-binding protein" refers to a protein having binding activity to immunoglobulin (or antibody or antibody fragment), and includes, for example, proteins containing one or more of the aforementioned immunoglobulin-binding domains. 【0019】In this specification, protein A (hereinafter also referred to as ProA) refers to protein A, a cell wall component of Staphylococcus aureus. Examples of immunoglobulin-binding domains of ProA include the B domain, C domain, D domain, A domain, E domain, and the Z domain, which is a modified version of the B domain. 【0020】 1. Immunoglobulin-binding protein The immunoglobulin-binding protein provided in the present invention is a mutant immunoglobulin-binding protein containing at least one mutant immunoglobulin-binding domain derived from the immunoglobulin-binding domain of ProA. The mutant immunoglobulin-binding domain can be obtained by adding a predetermined mutation to the immunoglobulin-binding domain derived from the parent domain ProA or a variant thereof. Hereinafter, the mutant immunoglobulin-binding domain provided in the present invention will also be referred to as the mutant immunoglobulin-binding domain of the present invention. 【0021】 Examples of parent domains for the mutant immunoglobulin-binding domains of the present invention include the B domain, Z domain, C domain of ProA, and their variants. 【0022】 The B domain of ProA is a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 1. A variant of the B domain is a polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 1 and possessing immunoglobulin-binding activity. The Z domain of ProA is a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 2. A variant of the Z domain is a polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and possessing immunoglobulin-binding activity. The C domain of ProA is a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 3. A variant of the C domain is a polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and possessing immunoglobulin-binding activity. 【0023】From the viewpoint of increasing protein expression levels in transformants (PNAS, 1989, 86:8247-8251, Fig. 2) and facilitating the creation of polynucleotides encoding immunoglobulin-binding proteins by linking multiple domains (WO2010 / 110288), the parent domain may include a substitution from Ala to Val at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 3. Furthermore, from the viewpoint of improving the chemical stability of the immunoglobulin-binding protein and increasing alkali resistance, the parent domain may further include a substitution from Gly to Ala at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 3 (Journal of Chromatography B, 2007, 848(1):40-47). Therefore, another example of the parent domain is an immunoglobulin-binding domain variant comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 3, where Val is located at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 3, or where Ala is located at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 3, or both. 【0024】 Mutants of the B, Z, or C domains used as the parent domain can be created by modifying the amino acid sequence of those domains, such as by inserting, substituting, or deleting amino acid residues, or by chemically modifying amino acid residues. Known methods for inserting, deleting, substituting, or deleting amino acid residues include site-specific mutation (SMT) of the polynucleotide encoding the domain. 【0025】The mutant immunoglobulin-binding domain of the present invention has the following mutations (a) and (b) introduced into the parent domain: (a) Substitution of an amino acid residue at the position corresponding to position 3 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Asp; (b) Insertion of at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to position 3 and position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. 【0026】 With respect to (a) above, the amino acid residue at the position corresponding to the 3rd position of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Asp, preferably Asn. The amino acid residue to be substituted is preferably Ala. In one embodiment, the mutation of (a) is the substitution of Asn at the position corresponding to the 3rd position of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Asp, preferably Ala. 【0027】With respect to (b) above, the inserted amino acid residue is preferably Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, or Tyr, more preferably at least one selected from the group consisting of Ala, Asp, Gln, Glu, and Leu, even more preferably Ala, Asp, Gln, Glu, or Leu, and even more preferably Ala or Leu. The amino acid residues at positions 3 and 4 of any of the amino acid sequences of SEQ ID NOs. 1 to 3 in the parent domain are preferably Asn and Lys, respectively, and preferably the parent domain does not have an amino acid residue between the position corresponding to position 3 and the position corresponding to position 4. Therefore, in one embodiment, the mutation in (b) is the insertion of the aforementioned amino acid residue between Asn at the position corresponding to position 3 and Lys at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs. 1 to 3. In another embodiment, if the mutation (a) or the later-described mutation (d) is introduced to the parent domain prior to the mutation (b), the amino acid residue at the position corresponding to the 3rd or 4th position may vary depending on the mutation (a) or (d). 【0028】Preferably, the mutant immunoglobulin-binding domain of the present invention has, in addition to the mutations in (a) and (b) above, at least one further mutation selected from the group consisting of (c) to (j) below introduced to the parent domain: (c) Substitution of an amino acid residue with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (d) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (e) Substitution of an amino acid residue with Ala or Asp at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (f) Substitution of an amino acid residue with Ala, Gln or Glu at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (g) Substitution of an amino acid residue with Ala at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (h) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (i) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. (j) Substitution of an amino acid residue with Ala or Arg at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. 【0029】 With respect to (c) above, the amino acid residue at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Val, preferably Ala. In one embodiment, the mutation in (c) is the substitution of Ala with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3. 【0030】 Regarding (d) above, the amino acid residue at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Arg, and is preferably Lys. The amino acid residue to be substituted is preferably Ala. In one embodiment, the mutation of (d) is the substitution of Lys at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg, preferably Ala. 【0031】 With respect to (e) above, the amino acid residue at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Asp, preferably Asn. In one embodiment, the mutation of (e) is the substitution of Asn at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Asp. 【0032】 Regarding (f) above, the amino acid residue at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala, Gln, or Glu, and is preferably Asn. In one embodiment, the mutation of (f) is the substitution of Lys at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala, Gln, or Glu. 【0033】 With respect to (g) above, the amino acid residue at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala, preferably Gly. In one embodiment, the mutation of (g) is the substitution of Gly at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala. 【0034】 Regarding (h) above, the amino acid residue at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Arg, preferably Lys. The amino acid residue to be substituted is preferably Arg. In one embodiment, the mutation of (h) is the substitution of Lys at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg, preferably Arg. 【0035】With respect to (i) above, the amino acid residue at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Arg, preferably Lys. The amino acid residue to be substituted is preferably Arg. In one embodiment, the mutation in (i) is the substitution of Lys at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg, preferably Arg. 【0036】 With respect to (j) above, the amino acid residue at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 in the parent domain is a residue other than Ala or Arg, preferably Lys. The amino acid residue to be substituted is preferably Arg. In one embodiment, the mutation of (j) is the substitution of Lys at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg, preferably Arg. 【0037】 Therefore, the parent domain of the mutant immunoglobulin-binding domain of the present invention preferably has Ala or Val at the position corresponding to position 1 (the same applies hereinafter to any amino acid sequence of SEQ ID NOs: 1 to 3), Asn at the position corresponding to position 3, Lys at the position corresponding to position 4, Asn at the position corresponding to position 6, Asn at the position corresponding to position 11, Gly or Ala at the position corresponding to position 29, and Lys at the positions corresponding to positions 49, 50 and 58, and does not have an amino acid residue between the position corresponding to position 3 and the position corresponding to position 4. 【0038】The mutant immunoglobulin-binding domain of the present invention is produced by introducing at least the mutations (a) and (b) into a parent domain consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 3 or an amino acid sequence having at least 80% identity thereto. Preferably, the mutant immunoglobulin-binding domain of the present invention is produced by introducing the mutations (a) and (b), as well as at least one mutation selected from the group consisting of (c) to (j), into the parent domain. In one embodiment, the mutant immunoglobulin-binding domain of the present invention is produced by introducing at least the mutations (a), (b), and (c) into the parent domain. Preferably, the mutant immunoglobulin-binding domain of the present invention is produced by introducing the mutations (a), (b), and (c), as well as at least one mutation selected from the group consisting of (d) to (j), into the parent domain. In another embodiment, the mutant immunoglobulin-binding domain of the present invention is produced by introducing at least the mutations (a), (b), (c), (e), and (g) into the parent domain. Preferably, the mutant immunoglobulin-binding domain of the present invention is produced by introducing the mutations (a), (b), (c), (e), and (g), as well as at least one mutation selected from the group consisting of (d), (f), (h), (i), and (j), into the parent domain. In another embodiment, the mutant immunoglobulin-binding domain of the present invention is produced by introducing the mutations (a), (b), (c), (e), (f), (g), and (h), into the parent domain. Preferably, the mutant immunoglobulin-binding domain of the present invention is produced by introducing the mutations (a), (b), (c), (e), (f), (g), and (h), as well as at least one mutation selected from the group consisting of (d), (i), and (j), into the parent domain. However, in the embodiments described above, if the parent domain already contains the mutations selected from (a) to (j), it is not necessary to reintroduce the mutations.For example, if the parent domain is a domain consisting of the amino acid sequence of SEQ ID NO: 2 into which the mutations (c) and (g) have been introduced, it is not necessary to reintroduce the mutations (c) and (g). 【0039】 One method for mutating the parent domain is to introduce a mutation into the polynucleotide encoding the parent domain so that a desired amino acid residue is substituted or inserted. Specific methods for introducing mutations into polynucleotides include site-directed mutation, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68), and the detailed procedures for these are well known to those skilled in the art. 【0040】 The mutant immunoglobulin-binding domain of the present invention obtained by the procedure described above may be a polypeptide chain having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 1 to 3, and having the following amino acid residues (A) and (B): (A) Ala or Asp, preferably Ala, at the position corresponding to position 3 of the amino acid sequence of any of SEQ ID NOs: 1 to 3. (B) The following amino acid residues between the position corresponding to the 3rd position and the position corresponding to the 4th position of any of the amino acid sequences of SEQ ID NOs: At least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr; preferably Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, or Tyr; more preferably Ala, Asp, Gln, Glu, or Leu; even more preferably Ala or Leu. 【0041】Preferably, the mutated immunoglobulin binding domain of the present invention has at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 3, and has the amino acid residues of (A) and (B) above, and at least one amino acid residue selected from the group consisting of the following (C) to (J), and may be a polypeptide chain consisting of an amino acid sequence. (C) Val at the position corresponding to the 1st position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (D) Ala or Arg, preferably Ala, at the position corresponding to the 4th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (E) Ala or Asp at the position corresponding to the 6th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (F) Ala, Gln or Glu at the position corresponding to the 11th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (G) Ala at the position corresponding to the 29th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (H) Ala or Arg, preferably Arg, at the position corresponding to the 49th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (I) Ala or Arg, preferably Arg, at the position corresponding to the 50th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. (J) Ala or Arg, preferably Arg, at the position corresponding to the 58th position of the amino acid sequence of any one of SEQ ID NOs: 1 to 3. 【0042】In one embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having at least the amino acid residues (A), (B), and (C). In a preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having the amino acid residues (A), (B), and (C), as well as at least one amino acid residue selected from the group consisting of (D) to (J). In another embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having at least the amino acid residues (A), (B), (C), (E), and (G). In a preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having the amino acid residues (A), (B), (C), (E), and (G), and at least one amino acid residue selected from the group consisting of (D), (F), (H), (I), and (J). In another embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having at least the amino acid residues (A), (B), (C), (E), (F), (G), and (H). In a preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with any of the amino acid sequences of SEQ ID NOs: 1 to 3, and having at least one amino acid residue selected from the group consisting of (A), (B), (C), (E), (F), (G), and (H), and (D), (I), and (J). 【0043】In one preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having at least the amino acid residues (A), (B), and (C). In a more preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having the amino acid residues (A), (B), and (C), as well as at least one amino acid residue selected from the group consisting of (D) to (J). In another preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having at least the amino acid residues (A), (B), (C), (E), and (G). In a more preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having the amino acid residues (A), (B), (C), (E), and (G), as well as at least one amino acid residue selected from the group consisting of (D), (F), (H), (I), and (J). In another preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having at least the amino acid residues (A), (B), (C), (E), (F), (G), and (H). In another preferred embodiment, the mutant immunoglobulin-binding domain of the present invention may be a polypeptide chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 and having the amino acid residues (A), (B), (C), (E), (F), (G), and (H), as well as at least one amino acid residue selected from the group consisting of (D), (I), and (J). 【0044】In a more preferred embodiment, the mutant immunoglobulin binding domain of the present invention may be a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 3, provided that it has the amino acid residues of (A), (B), (C), (E), (F), (G) and (H). In another preferred embodiment, the mutant immunoglobulin binding domain of the present invention may be a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 3, provided that it has the amino acid residues of (A), (B), (C), (E), (F), (G) and (H), and at least one amino acid residue selected from the group consisting of (D), (I) and (J). 【0045】 The mutant immunoglobulin binding domain of the present invention has improved alkali resistance and is less likely to dissociate from a carrier as compared with the parent domain, specifically, a domain consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 3 (for example, SEQ ID NO: 3). Further, the mutant immunoglobulin binding domain of the present invention is less likely to be decomposed in a culture when the domain is produced by cell culture or the like, as compared with the parent domain, specifically, a domain consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 3 (for example, SEQ ID NO: 3), and thus can be produced in a higher yield. Furthermore, as shown in the examples described later, the mutant immunoglobulin binding domain of the present invention also has improved alkali resistance, is less likely to dissociate from a carrier, and is less likely to be decomposed in a culture, as compared with a mutant domain obtained by adding either one of the mutations of (a) and (b) to the amino acid sequence of the parent domain, specifically, any one of SEQ ID NOs: 1 to 3 (for example, SEQ ID NO: 3). Therefore, the mutant immunoglobulin binding domain of the present invention can be suitably used as an affinity ligand. 【0046】 The mutant immunoglobulin binding domain of the present invention may have mutations other than (a) to (j) as long as the effect of improving alkali resistance or suppressing dissociation from a carrier is not lost, and as long as the immunoglobulin binding activity is not lost. 【0047】The immunoglobulin-binding protein of the present invention may contain one or more of the mutant immunoglobulin-binding domains described above. Preferably, the immunoglobulin-binding protein of the present invention contains two or more, more preferably three or more, and even more preferably four or more of the mutant immunoglobulin-binding domains. On the other hand, the immunoglobulin-binding protein of the present invention contains preferably 12 or fewer, more preferably 8 or fewer, and even more preferably 6 or fewer of the mutant immunoglobulin-binding domains. For example, the immunoglobulin-binding protein of the present invention contains preferably 2 to 12, more preferably 3 to 8, and even more preferably 4 to 6 of the mutant immunoglobulin-binding domains. Immunoglobulin-binding proteins containing such a number of mutant immunoglobulin-binding domains have particularly improved alkali resistance, are less likely to dissociate from the carrier, and are less likely to degrade in the culture medium. When the immunoglobulin-binding protein of the present invention contains two or more mutant immunoglobulin-binding domains of the present invention, these mutant immunoglobulin-binding domains may be of the same type or different types, but are preferably of the same type. 【0048】 The immunoglobulin-binding protein of the present invention may contain other immunoglobulin-binding domains other than the mutant immunoglobulin-binding domain of the present invention described above. Examples of such other domains include the native ProA immunoglobulin-binding domain (e.g., the B, Z, or C domain of ProA) and their variants other than the mutant immunoglobulin-binding domain of the present invention. The total number of immunoglobulin-binding domains in such an immunoglobulin-binding protein containing the mutant immunoglobulin-binding domain of the present invention and other immunoglobulin-binding domains (e.g., the native ProA immunoglobulin-binding domain) is preferably 2 to 12, more preferably 3 to 8, and even more preferably 4 to 6, of which the number of mutant immunoglobulin-binding domains of the present invention is preferably 10% to 60% and more preferably 20% to 50% of the total number of immunoglobulin-binding domains. 【0049】From the viewpoint of increasing the amount of immunoglobulin-binding protein immobilized on a carrier, increasing the number of binding sites to the carrier, and increasing the antibody binding capacity, any amino acid residue or peptide may be added or inserted at one or more locations, either at the N-terminus, C-terminus, or between domains, of the immunoglobulin-binding domain contained in the immunoglobulin-binding protein of the present invention. Preferred examples of the amino acid residue or peptide to be added or inserted include Cys, Lys, Pro, (Pro) ｐ , (Ala-Pro) ｑ , and (Glu-Ala-Ala-Ala-Lys) ｒ (Examples include p being an integer between 2 and 300, preferably between 12 and 24; q being an integer of 4 or more, preferably between 4 and 10; and r being an integer of 2 or more, preferably between 2 and 6.) 【0050】2. Production of Immunoglobulin-Binding Proteins The immunoglobulin-binding proteins of the present invention can be produced by methods known in the art, such as chemical synthesis methods based on amino acid sequences or recombinant methods. For example, the immunoglobulin-binding proteins of the present invention can be produced using known genetic recombination techniques described in Current Protocols In Molecular Biology by Frederick M. Ausbel et al. or Molecular Cloning (Cold Spring Harbor Laboratory Press, 3rd edition, 2001) edited by Sambrook et al. That is, by transforming a host such as E. coli with an expression vector containing a polynucleotide encoding the immunoglobulin-binding protein of the present invention, and culturing the resulting recombinant in an appropriate liquid medium, the target protein can be obtained in large quantities and economically from the cultured cells. Preferred expression vectors include any known vector that can replicate in host cells, such as the plasmid described in U.S. Patent No. 5,151,350 and the plasmid described in Molecular Cloning edited by Sambrook et al., as mentioned above. The host for transformation is not particularly limited, but known hosts used to express recombinant proteins, such as bacteria like Escherichia coli, fungi, insect cells, and mammalian cells, can be used. Any method known in the art to transform the host by introducing nucleic acids into it may be used depending on the host, such as the known method described in Molecular Cloning edited by Sambrook et al., as mentioned above. Methods for culturing the resulting transformant (preferably a microbial cell such as bacteria) and recovering the expressed protein are well known to those skilled in the art. Alternatively, the immunoglobulin-binding protein of the present invention may be expressed using a cell-free protein synthesis system. 【0051】Accordingly, the present invention also provides polynucleotides (such as DNA) encoding the immunoglobulin-binding proteins of the present invention, vectors containing the same, and transformants containing the same. 【0052】 3. Affinity Carrier The immunoglobulin-binding protein of the present invention can be used as an affinity ligand. By immobilizing the immunoglobulin-binding protein of the present invention on a solid-phase carrier, an affinity carrier containing the immunoglobulin-binding protein of the present invention as a ligand (hereinafter also referred to as the affinity carrier of the present invention) can be produced. Therefore, the affinity carrier of the present invention comprises a solid-phase carrier and the immunoglobulin-binding protein of the present invention immobilized on the solid-phase carrier. 【0053】 The affinity carrier of the present invention has the advantage of improved alkali resistance and suppressed ligand leakage. That is, compared to an affinity carrier that uses an immunoglobulin-binding protein as a ligand that includes a domain consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 3 (e.g., SEQ ID NO: 3) instead of the mutant immunoglobulin-binding domain of the present invention, the affinity carrier of the present invention has improved alkali resistance and suppressed ligand leakage. Furthermore, as shown in the examples described below, the affinity carrier of the present invention has improved alkali resistance and suppressed ligand leakage compared to an affinity carrier that uses an immunoglobulin-binding protein as a ligand that includes a mutant domain in which either (a) or (b) of the above mutation has been added to the amino acid sequence of any of SEQ ID NOs: 1 to 3 (e.g., SEQ ID NO: 3) instead of the mutant immunoglobulin-binding domain of the present invention. 【0054】The affinity carrier of the present invention can be used as a packed bed or as a suspension. The suspension includes what is known as a fluidized bed and a pure suspension, in which particles can move freely. In the case of monoliths, packed beds, and fluidized beds, the separation procedure generally follows conventional chromatography methods using a concentration gradient. In the case of pure suspensions, a batch method is used. 【0055】 Preferably, the affinity carrier of the present invention is a carrier for affinity chromatography. In one embodiment, the affinity carrier of the present invention is a packing material. In another embodiment, the affinity carrier of the present invention may be in the form of a column, tip, capillary or filter packed with a solid-phase carrier on which ligands are immobilized. 【0056】 The solid-phase supports included in the affinity support of the present invention include organic supports such as synthetic polymer supports and natural polymer supports; inorganic supports; and organic-organic composite supports and organic-inorganic composite supports that combine these. Examples of synthetic polymer supports include those composed of polyvinyl alcohols, poly(meth)acrylates, poly(meth)acrylamides, polystyrenes, and ethylene-maleic anhydride copolymers. Examples of natural polymer supports include those composed of polysaccharides such as agarose, dextran, mannan, and cellulose. These may also be physically crosslinked or chemically crosslinked. Examples of inorganic supports include those composed of glass beads, silica gel, metals, and metal oxides. Among these, synthetic polymer supports are preferred from the viewpoint of flow velocity characteristics. 【0057】 A preferred example of the synthetic polymer support is a copolymer of a monofunctional unsaturated monomer and a polyfunctional unsaturated monomer. The monofunctional unsaturated monomer is preferably a monofunctional unsaturated monomer having an epoxy group or a ring-opening epoxy group. The amount of the monofunctional unsaturated monomer and polyfunctional unsaturated monomer used is typically 1 to 100 parts by mass, preferably 1 to 50 parts by mass, of the polyfunctional unsaturated monomer per 100 parts by mass of the monofunctional unsaturated monomer. 【0058】 The solid phase support can take any shape, such as particulate, monolithic, plate-like, chip-like, fibrous, or film-like (including hollow fibers). However, from the viewpoint of target substance capture characteristics, particulate, monolithic, plate-like, fibrous, or film-like forms are preferred, with particulate being more preferred. The solid phase support may be non-porous or porous, but it is preferable that it be porous. Preferably, the solid phase support is porous particles. Furthermore, the solid phase support is preferably water-insoluble. 【0059】 The particle size of the solid-phase support is preferably 30 μm or larger from the viewpoint of flow velocity characteristics, and preferably 300 μm or smaller from the viewpoint of target substance capture characteristics. Such particle size can be adjusted by the conditions during polymerization and classification. In this specification, "particle size" refers to the volume-average particle diameter obtained by a laser diffraction scattering particle size distribution analyzer. 【0060】 When the solid phase support is porous particles, its specific surface area is the specific surface area in the pore size range of 10 nm to 5000 nm, when measuring the pores corresponding to the pore diameter range of 10 nm to 5000 nm, preferably 70 m². 2 It is 1 / g or more, and more preferably 90m 2 It is 150 m or more per gram, and preferably 150 m 2 It is less than or equal to / g. In this specification, "specific surface area" refers to the value obtained by dividing the surface area of ​​pores with a diameter of 10 to 5000 nm, obtained by a mercury porosimeter, by the dry mass of the particles. 【0061】 In one embodiment, the solid-phase support preferably has a volume-average pore diameter of 100 to 500 nm. For example, if the support is a synthetic polymer, the volume-average pore diameter is preferably 100 to 400 nm, and more preferably 200 to 300 nm. In this specification, "volume-average pore diameter" refers to the volume-average pore diameter of pores with a diameter of 10 to 5000 nm obtained by a mercury porosimeter. 【0062】The solid phase support may be a commercially available product or one synthesized according to conventional methods. An example of porous particles used as the solid phase support is a porous crosslinked particle (WO2019 / 039545) obtained by polymerizing divinylbenzene, ethylvinylbenzene, and glycidyl methacrylate, and further crosslinking with adipic acid dihydrazide. 【0063】 The ligand (immunoglobulin-binding protein) can be attached to the solid support using a general method for immobilizing the protein on a support. Examples include: using a support having a carboxyl group and activating the carboxyl group with N-hydroxysuccinimide to react with the amino group of the ligand; using a support having an amino group or a carboxyl group and reacting it with the carboxyl group or amino group of the ligand in the presence of a dehydrating condensation agent such as a water-soluble carbodiimide to form an amide bond; using a support having a hydroxyl group and activating it with a cyanogen halide such as cyanogen bromide to react with the amino group of the ligand; tosylating or tresyling the hydroxyl group of the support and reacting it with the amino group of the ligand; introducing an epoxy group into the support using bis-epoxide, epichlorohydrin, etc., and reacting it with the amino group, hydroxyl group, or thiol group of the ligand; and using a support having an epoxy group to react it with the amino group, hydroxyl group, or thiol group of the ligand. Of the above, from the viewpoint of stability in the aqueous solution in which the reaction is carried out, the method of attaching the ligand via an epoxy group is desirable. 【0064】Alcoholic hydroxyl groups, which are ring-opened epoxy groups formed by the ring-opening of epoxy groups, hydrophilize the support surface, preventing non-specific adsorption of proteins and other substances, and improve the toughness of the support in water, thus preventing the support from breaking down under high flow rates. Therefore, if there are residual epoxy groups in the support that are not bound to the ligand after the ligand has been immobilized, it is preferable to ring-open these residual epoxy groups. Methods for ring-opening epoxy groups in a support include, for example, heating or stirring the support in an aqueous solvent containing an acid or alkali at room temperature. Alternatively, the epoxy groups may be ring-opened using blocking agents having mercapto groups, such as mercaptoethanol and thioglycerol, or blocking agents having amino groups, such as monoethanolamine. More preferable are ring-opened epoxy groups obtained by ring-opening the epoxy groups contained in the support with thioglycerol. Thioglycerol has advantages such as lower toxicity as a raw material compared to mercaptoethanol, and the epoxy ring-opening group to which thioglycerol is added exhibits lower non-specific adsorption and higher dynamic bonding capacity compared to ring-opening groups formed by blocking agents containing amino groups. 【0065】 If necessary, molecules of any length (spacers) may be introduced between the solid support and the ligand. Examples of such spacers include polymethylene chains, polyethylene glycol chains, and sugar chains. 【0066】 4. Method for Isolating Antibodies or Fragments thereof A method for isolating antibodies or fragments thereof using the affinity carrier of the present invention (hereinafter also referred to as the isolation method of the present invention), according to one embodiment of the present invention, will be described. The isolation method of the present invention preferably includes the steps of: passing a sample containing a target substance (an antibody or fragment thereof as defined above) through the affinity carrier of the present invention to bind the target substance to the carrier (step 1); and recovering the target substance from the carrier (step 2). 【0067】In step 1, a sample containing an antibody or a fragment thereof as a target substance is passed through a column packed with the affinity carrier of the present invention under conditions that the target substance binds to the ligand (the immunoglobulin-binding protein of the present invention). In this step, most of the substances in the sample other than the target substance pass through the column without binding to the ligand. After this, if necessary, the carrier may be washed with a washing solution to remove some of the substances weakly retained by the ligand. The washing solution can be a neutral buffer containing a salt such as NaCl, for example, an aqueous solution of sodium phosphate / sodium chloride, a sodium dihydrogen phosphate / disodium hydrogen phosphate solution, a citrate / disodium hydrogen phosphate solution, a hydrochloric acid / tris(hydroxymethyl)aminomethane solution, or a HEPES / sodium hydroxide solution. 【0068】 In step 2, an acidic eluent is flowed through the carrier to elute the target substance bound to the ligand, thereby recovering the purified target substance. For example, a sodium acetate solution can be used as the eluent. The pH of the eluent is preferably 2 or higher, more preferably 2.5 or higher, even more preferably 3 or higher, while preferably 5 or lower, more preferably 4.5 or lower, and even more preferably 4 or lower. For example, the pH of the eluent is preferably 2 to 5, more preferably 2 to 4.5, more preferably 2 to 4, even more preferably 2.5 to 5, even more preferably 2.5 to 4.5, even more preferably 2.5 to 4, even more preferably 3 to 5, even more preferably 3 to 4.5, and even more preferably 3 to 4. 【0069】 The target substance contained in the eluate obtained in step 2 may be further purified. The purification of the target substance can be carried out, for example, by using cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, hydrophilic interaction chromatography, hydrophobic interaction chromatography, size exclusion chromatography, etc., individually or in appropriate combinations. 【0070】The isolation method of the present invention may further include a step (step 3) of passing an alkaline solution through the affinity carrier after step 2. In step 3, the affinity carrier is washed with the alkaline solution (CIP washing). Examples of the alkaline solution used in step 3 include aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous triethylamine solution, and tetrabutylammonium hydroxide. The molar concentration of the alkali salt in the alkaline solution used in step 3 is preferably 0.01 to 4.0 M, more preferably 0.1 to 2.0 M or more. The pH of the alkaline solution used is preferably pH 11.0 to 15.0, more preferably pH 12.0 to 14.0. 【0071】 After step 3, the affinity carrier can be used again to isolate the target substance. Therefore, in one embodiment, the isolation method of the present invention includes repeating steps 1 to 3 multiple times, preferably 50 times or more, and more preferably 100 times or more. Because the affinity carrier of the present invention has high alkali resistance, it can maintain a high dynamic binding capacity (DBC) to the target substance even after repeated use. Furthermore, because ligand leakage is suppressed in the chromatography carrier of the present invention, the purity of the isolated target substance can be maintained even after repeated use. 【0072】 The present invention will be described in more detail below with reference to examples. Furthermore, the following description provides a general overview of the aspects of the present invention, and the present invention is not limited by such description without particular reason. 【0073】 (Preparation Example) Ligand (Immunoglobulin-binding protein) Preparation Immunoglobulin-binding proteins PrA-1 to PrA-20 were obtained. PrA-1 to PrA-20 are immunoglobulin-binding proteins in which mutant immunoglobulin-binding domains, in which the mutations described in Table 1 have been introduced into the parent domain, are linked in series. 【0074】 【0075】The expression and purification of PrA-1 to PrA-20 were performed as follows: Escherichia coli BL21 (DE3) was transformed using plasmids encoding PrA-1 to PrA-20, and the resulting transformants were cultured in a nutrient-rich medium at 37°C until the logarithmic growth phase. Subsequently, 1 mM isopropyl-β-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added to the medium, and the cells were cultured at 37°C for 4 hours to express the target proteins. The culture medium was then centrifuged to remove the supernatant, and the resulting cells were lysed by adding 30 mM Tris buffer at pH 9.5 containing oval-derived lysozyme (Wako Pure Chemical Industries, Ltd.) and polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical Industries, Ltd.). Recombinant immunoglobulin-binding proteins were purified from the obtained cell lysates by cation exchange chromatography (SP-Sepharose FF, GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharose FF, GE Healthcare Biosciences). The purified immunoglobulin-binding proteins were dialyzed against 10 mM citrate buffer (pH 6.0). The purity of the recombinant immunoglobulin-binding proteins, as confirmed by SDS-PAGE, was over 95%. 【0076】 (Example 1) Preparation of affinity carrier (1) Synthesis of porous particles 2.69 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the polyvinyl alcohol was dissolved by heating and stirring to obtain an aqueous solution S. A monomer composition consisting of 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Ltd.), and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Ltd.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. 【0077】The entire aqueous solution S was poured into a separable flask, a thermometer, a stirring blade, and a condenser were attached, and the flask was placed in a hot water bath and stirred under a nitrogen atmosphere. The entire monomer solution was poured into the separable flask and heated in a hot water bath. When the internal temperature reached 85°C, 1.34 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to adjust the internal temperature to 86°C. The solution was then stirred for 3 hours while maintaining the temperature at 86°C. After the resulting reaction solution was cooled, it was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water to obtain a dispersion of porous particles with a particle concentration of 10% by mass. The porous particles contained in this dispersion are referred to as "porous particles 1". 【0078】 To 100 g of the dispersion of porous particles 1, 0.956 g of dihydrazide adipic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), 8 g of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.), and 1.418 g of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was heated to 70°C and stirred for 8 hours while maintaining the temperature. After cooling the resulting reaction solution, it was filtered and washed with pure water and ethanol. Next, the particles were dispersed in pure water to a particle concentration of 10% by mass to obtain a dispersion of porous particles. The porous particles contained in this dispersion are referred to as "porous particles 2". 【0079】 Ethylene glycol diglycidyl ether was reacted with the thioglycerol-derived hydroxyl groups contained in porous particles 2. 8.7 g of pure water, 1.2 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.10 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 11.2). 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810, manufactured by Nagase ChemteX Corporation) and 8 mL of a dispersion of porous particles 2 were added to the carbonate buffer, and the mixture was shaken at 23°C for 16 hours. Next, the particles were dispersed in pure water to a particle concentration of 50% by volume to obtain a dispersion of porous particles. The porous particles contained in this dispersion are referred to as "porous particles 3". 【0080】(2) Washing of porous particles The porous particles 3 were washed. Pure water was filtered out of 16 mL of the dispersion of porous particles 3, and the solids were allowed to settle at the bottom of the container to form a bed. 8 mL of 0.5 M sodium hydroxide aqueous solution was poured onto the bed from above to below, and filtered without stirring (hereinafter also referred to as "cut-out washing") was performed twice. Subsequently, 8 mL of 0.5 M sodium hydroxide aqueous solution was added to the bed, the entire container was stirred, and then filtered (hereinafter also referred to as "reslurry washing") was performed once. Next, the particles were dispersed in pure water so that the particle concentration was 50 volume%, and a dispersion of porous particles was obtained. The porous particles contained in this dispersion are referred to as "porous particles 4". 【0081】 (3) Ligand immobilization Ligands were immobilized on porous particles 4. Specifically, 28.8 g of pure water, 5.4 g of sodium sulfate (Wako Pure Chemical Industries, Ltd.), 0.2 g of sodium bicarbonate (Wako Pure Chemical Industries, Ltd.), and 0.16 g of sodium carbonate (Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 9.3). 0.17 g of the immunoglobulin-binding protein PrA-1 prepared in the preparation example and 8 mL of porous particles 4 were added to 25 mL of the carbonate buffer, and the mixture was shaken at 23°C for 1.5 hours. The resulting reaction solution was filtered and the particles were recovered. 8.8 g of pure water, 0.1 g of sodium sulfate (Wako Pure Chemical Industries, Ltd.), and 0.03 g of sodium hydroxide (Wako Pure Chemical Industries, Ltd.) were mixed to obtain a buffer, and then 4.5 g of thioglycerol (Tokyo Chemical Industries, Ltd.) was added to prepare a hydrophilization reaction solution. The hydrophilization reaction solution was added to the aforementioned particles, and the mixture was shaken and stirred at 23°C for 16 hours to carry out the hydrophilization reaction. Next, the particles were dispersed in pure water to a particle concentration of 50% by volume to obtain a dispersion of ligand-immobilized porous particles. The ligand-immobilized porous particles contained in this dispersion are referred to as "porous particles 5". 【0082】(4) Washing of Ligand-Immobilized Porous Particles The porous particles 5 were washed. Pure water was filtered out of 16 mL of the dispersion of porous particles 5, and the remaining solid bed was subjected to two cut-out washes with 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4), followed by one reslurry wash with 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4), and then filtered wash with sodium citrate buffer. Next, the particles were dispersed in pure water to a particle concentration of 50 vol%, and a dispersion of ligand-immobilized porous particles was obtained. The ligand-immobilized porous particles contained in this dispersion are referred to as "carrier 1". 【0083】 (Examples 2-17) Carriers 2-17 were prepared by performing the same procedure as in Example 1, except that the immunoglobulin-binding proteins used in Example 1(3) were changed to PrA-2-PrA-17, respectively. 【0084】 (Comparative Examples 1-3) The carriers for Comparative Examples 1-3 (carrier 18-carrier 20) were prepared by performing the same procedure as in Example 1, except that the immunoglobulin-binding proteins used in Example 1(3) were changed to PrA-18-PrA-20, respectively. 【0085】 (Test Example 1) Dynamic Binding Capacity (DBC) Measurement: The DBC of the support molecules in the examples and comparative examples was measured for the target protein (human IgG antibody, LGC 1875-0007) using a chromatography system (Cytiva AKTA avant25) with a retention time of 4 minutes. The support molecules were packed into a 4 mL column (5 mm diameter × 200 mm length). The sample solution used was a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) in which the target protein was dissolved at 5 mg / mL. The sample solution was delivered to the column with a retention time of 4 minutes, and the DBC (mg / mL) was determined from the amount of protein captured at the 10% breakthrough at the elution tip and the column packing volume. The DBC of each support molecule was evaluated according to the following criteria. A: 62 or higher (mg / mL) B+: 60 or higher but less than 62 (mg / mL) B: 58 or higher but less than 60 (mg / mL) C: Less than 58 (mg / mL) 【0086】(Test Example 2) Alkali Resistance Test The carrier-packed column used in Test Example 1 was set in an AKTA avant25 (Cytiva), and 20 mL of 0.5 M sodium hydroxide was flowed through the column. After the solution was delivered, the column was left at room temperature for 24 hours, and then the DBC was measured using the same procedure as in Test Example 1. The ratio (%) of DBC after treatment to DBC before treatment with 0.5 M sodium hydroxide was determined as alkali resistance. The alkali resistance of each carrier was evaluated according to the following criteria: A: 95% or more B+: 93% or more to less than 95% B: 90% or more to less than 93% C: Less than 90% 【0087】 (Test Example 3) Protein A Leakage Measurement Test Using a chromatography system (AKTA avant25, manufactured by Cytiva), 7.5 mL of cell culture medium (containing Herceptin: 5.21 mg / mL) was loaded onto a 0.8 mL column (5 mmφ × 40 mm long) packed with one of the carriers from Examples 1-17 and Comparative Examples 1-3. After a retention time of 4 minutes, the column was washed, and then the antibody was eluted and recovered. A 20 mM sodium phosphate / 500 mM sodium chloride aqueous solution (pH 7.5) was used for washing, and a 100 mM sodium acetate aqueous solution (pH 3.3) was used as the eluent for antibody elution. After elution, the column was washed with 0.5 M sodium hydroxide and then re-equilibriumated with a 20 mM sodium phosphate / 500 mM sodium chloride aqueous solution (pH 7.5). The ligand protein content (g-IgG) in the obtained eluate was measured using a Protein A ELISA kit (F740), and the antibody content (μg-PrA) in the eluate was determined from the absorbance. The ligand protein leach (μg-PrA / g-IgG) per unit of antibody in the eluate was calculated. The leach for each carrier was evaluated according to the following criteria: A: less than 5 (μg-PrA / g-IgG) B+: 5 or more but less than 10 (μg-PrA / g-IgG) B: 10 or more but less than 15 (μg-PrA / g-IgG) C: 15 or more (μg-PrA / g-IgG) 【0088】For Examples 1, 2, 4, 13 and Comparative Example 3, the process from loading the cell culture medium to re-equilibriumizing the column was considered one cycle, and the same procedure was performed for 100 cycles. The amount of ligand protein leakage (leach) at the 100th cycle was calculated and evaluated in the same manner as above. 【0089】 (Test Example 4) Degradation resistance test to cell culture medium To remove antibodies from cell culture medium (containing Herceptin: 5.21 mg / mL), the cell culture medium was treated with protein A solution (Amsphere TM A3 (manufactured by JSR Corporation) was contacted for 1 hour, and the supernatant was collected by centrifugation to prepare an antibody-free culture supernatant. The obtained culture supernatant was mixed in equal volumes with immunoglobulin-binding proteins (PrA-1 to PrA-20, each at 2 mg / mL) and contacted at 37°C for 100 hours. The reaction solution was mixed in equal volumes with 2×Laemmli Sample Buffer containing 0.1 M DTT and electrophoresis was performed using an SDS polyacrylamide gel. The gel after electrophoresis was stained with Oriole stain and observed using a gel scanner. The intensity of the immunoglobulin-binding protein staining bands on the observed gel was quantified, and the ratio (%) of the immunoglobulin-binding protein band intensity after contact with the culture supernatant to before contact was calculated as the degradation resistance. The degradation resistance of each carrier was evaluated according to the following criteria. A: 85% or higher B+: 70% or higher but less than 85% B: 55% or higher but less than 70% C: Less than 55% 【0090】 The results for Test Examples 1-4 are shown in Tables 2-4 (in the table, n.d. means no data). 【0091】 【0092】 【0093】

Claims

1. An affinity carrier comprising a solid support and an immunoglobulin-binding protein bound to the solid support, wherein the immunoglobulin-binding protein comprises at least one mutant immunoglobulin-binding domain, the mutant immunoglobulin-binding domain consists of an amino acid sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 1 to 3, and the mutant immunoglobulin-binding domain has the following mutations: (a) substitution of an amino acid residue at the position corresponding to the 3rd position of the amino acid sequence of any of SEQ ID NOs: 1 to 3 with Ala or Asp; (b) insertion of at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to the 3rd and 4th positions of the amino acid sequence of any of SEQ ID NOs: 1 to 3. Affinity carrier.

2. The mutant immunoglobulin-binding domain further has at least one mutation selected from the group consisting of (c) to (j) below: (c) substitution of an amino acid residue with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (d) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (e) substitution of an amino acid residue with Ala or Asp at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (f) substitution of an amino acid residue with Ala, Gln or Glu at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (g) substitution of an amino acid residue with Ala at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (h) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (i) Substitution of an amino acid residue at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg; (j) Substitution of an amino acid residue at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg; The affinity carrier according to claim 1.

3. The affinity carrier according to claim 2, wherein the mutant immunoglobulin-binding domain has the mutations of (a), (b), (c), (e), and (g).

4. The affinity carrier according to claim 3, wherein the mutant immunoglobulin-binding domain has at least one mutation selected from the group consisting of the mutations (a), (b), (c), (e), and (g), and (d), (f), (h), (i), and (j).

5. The affinity carrier according to claim 1, wherein the immunoglobulin-binding protein comprises 2 to 12 immunoglobulin-binding domains.

6. An immunoglobulin-binding protein comprising at least one mutant immunoglobulin-binding domain, wherein the mutant immunoglobulin-binding domain consists of an amino acid sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 1 to 3, and the mutant immunoglobulin-binding domain has the following mutations: (a) substitution of an amino acid residue at the position corresponding to position 3 of the amino acid sequence of any of SEQ ID NOs: 1 to 3 with Ala or Asp; (b) insertion of at least one amino acid residue selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp, and Tyr between the position corresponding to position 3 and position 4 of the amino acid sequence of any of SEQ ID NOs: 1 to 3.

7. The mutant immunoglobulin-binding domain further has at least one mutation selected from the group consisting of (c) to (j) below: (c) substitution of an amino acid residue with Val at the position corresponding to position 1 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (d) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 4 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (e) substitution of an amino acid residue with Ala or Asp at the position corresponding to position 6 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (f) substitution of an amino acid residue with Ala, Gln or Glu at the position corresponding to position 11 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (g) substitution of an amino acid residue with Ala at the position corresponding to position 29 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (h) substitution of an amino acid residue with Ala or Arg at the position corresponding to position 49 of any of the amino acid sequences of SEQ ID NOs: 1 to 3; (i) Substitution of an amino acid residue at the position corresponding to position 50 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg; (j) Substitution of an amino acid residue at the position corresponding to position 58 of any of the amino acid sequences of SEQ ID NOs: 1 to 3 with Ala or Arg; The immunoglobulin-binding protein according to claim 6.

8. The immunoglobulin-binding protein according to claim 7, wherein the mutant immunoglobulin-binding domain has the mutations of (a), (b), (c), (e), and (g).

9. The immunoglobulin-binding protein according to claim 8, wherein the mutant immunoglobulin-binding domain has at least one mutation selected from the group consisting of the mutations (a), (b), (c), (e), and (g), and (d), (f), (h), (i), and (j).

10. A polynucleotide encoding an immunoglobulin-binding protein according to any one of claims 6 to 9.

11. A transformant into which the polynucleotide described in claim 10 has been introduced.

12. A method for isolating an antibody or a fragment thereof, comprising: (Step 1) passing a solution containing an antibody or a fragment thereof as a target substance through a chromatography carrier according to any one of claims 1 to 5 to bind the target substance to the carrier; (Step 2) recovering the target substance from the carrier; and (Step 3) passing an alkaline solution through the carrier.

13. The method according to claim 12, comprising repeating steps 1 to 3 multiple times.

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