WO2019039602A1 - Protein, and isolation method for antibodies or fragments thereof using carrier including said protein - Google Patents

Abstract

Provided is a carrier for affinity chromatography having high immunoglobulin purification efficiency. This protein contains two or more domains linked together by a linker. The domains have affinity to immunoglobulins. The linker is a peptide having a rod-shaped structure, or the linker includes (a) a linker comprising a polypeptide containing at least one proline and/or (b) a linker comprising a polypeptide including at least one peptide unit comprising Xa-(A)i-Xb or Xb-(A)i-Xa (where Xa represents an acidic amino acid, Xb represents a basic amino acid, A represents alanine, and i represents an integer of 3 or 4).

Description

Protein and method for isolating antibody or fragment thereof using carrier containing said protein

The present invention relates to a protein available as an affinity chromatography ligand, and a method for isolating an antibody or a fragment thereof using a carrier containing the protein.

In affinity chromatography, a carrier on which a substance (ligand) that specifically binds to a target substance to be separated is immobilized is used. Staphylococcus aureus protein A (SpA) and its variants are proteins that bind specifically to immunoglobulins and are one of the representative affinity chromatography ligands. Natural SpA contains five domains of E, D, A, B and C in order from the N-terminus as immunoglobulin binding domains. These domains, and Z domains, which are modified domains of B domains, are commonly used as affinity chromatography ligands. Furthermore, for the purpose of enhancing the immunoglobulin binding ability of the affinity chromatography carrier, it is also common to use a plurality of linked domains described above as a ligand.

When multiple proteins are artificially linked, short peptide sequences called spacers or linkers may be inserted between the proteins. Spacers or linkers are used to maintain the conformation and activity of the linked protein complex. In Non-Patent Document 1, in the interferon γ (INF-γ) -gp120 fusion protein, an alanine-proline linker of 10 to 34 residues is inserted between two proteins, and IFN-γ of the fusion protein The activity is described to be low when the linker length is short, but increased to 88% of the original when the linker length is 34 residues. In Non-Patent Document 2, insertion of a linker between two proteins of human serum albumin-IFN-α2b fusion protein improves the yield and stability of the fusion protein, as compared to a fusion protein without a linker. It is described that the antiviral activity was increased, and that the length of the linker was sufficient at 5 residues. In Patent Document 1, a protein in which a plurality of domains, each of which binds to the Fc region of an immunoglobulin, is linked by a linker consisting of GGSGGS repeat units, maintains affinity for immunoglobulin in the neutral region during affinity purification. It is described that immunoglobulins can be dissociated in weakly acidic regions.

International Publication No. 2015/050153

The increase in the number of immunoglobulin binding domains on the affinity chromatography support was not necessarily linked to the proportional increase in the immunoglobulin binding capacity or purification efficiency of the support. It is presumed that this is due to the interference between a plurality of linked domains and the effect of immunoglobulin bound to a ligand that the binding between the domain and the immunoglobulin is inhibited. The present invention relates to an affinity chromatography ligand having a high ability to bind to immunoglobulin and a carrier for affinity chromatography having high immunoglobulin purification efficiency using the same.

Accordingly, the present invention provides the following.
[1] A protein comprising two or more domains linked by a linker, wherein the domains have an affinity for immunoglobulin, and the linker is a peptide having a rod-like structure.
[2] A protein comprising two or more domains linked by a linker,
The domain has an affinity for immunoglobulins and
The linker, a linker comprising a polypeptide comprising at least one proline (a), and _{^{X a - (A) i -X}} b or ^X b - at least one _(A) consisting of i -X ^a peptide units Containing at least one of the following linkers (b) (wherein X ^a is an acidic amino acid, X ^b is a basic amino acid, A is alanine and i is an integer of 3 or 4): ,
protein.
[3] The protein according to [2], wherein the linker (a) comprises a polypeptide having a length of 8 amino acids or more.
[4] The protein according to [3], wherein the linker (a) contains 4 or more in total of peptide units selected from the group consisting of Pro-Xaa and Xaa-Pro.
[5] The protein according to [4], wherein Xaa is an amino acid other than Pro.
[6] The protein according to [4], wherein Xaa is Ala.
[7] The protein according to [4], wherein the linker consists of 4 or more Ala-Pro units.
[8] The protein according to [2], wherein the linker (b) comprises a polypeptide having a length of 10 amino acids or more.
[9] The linker (b) is Glu- (Ala) _i- Arg, Glu- (Ala) _i- Lys, Asp- (Ala) _i- Arg, Asp- (Ala) _i- Lys, Arg- (Ala) ) _{i -Glu,} including Lys- (Ala) i -Glu, Arg- (Ala) i -Asp, and Lys- _(Ala) i 2 or more in total peptide units selected from the group consisting -Asp, [ 2] The described protein.
[10] The protein according to [9], wherein the peptide unit is Glu- (Ala) ₃ -Lys or Lys- (Ala) ₃ -Glu.
[11] The N-terminus of the linker is linked to the C-terminus of any one of the two or more domains, and the C-terminus of the linker is linked to the N-terminus of another domain, [1] to [10] The protein according to any one of
[12] The protein according to any one of [1] to [11], wherein each of the two or more domains is an immunoglobulin binding domain of protein A or protein L or a variant thereof.
[13] A polypeptide, wherein each of the two or more domains consists of the amino acid sequence shown in any of SEQ ID NOs: 3 to 10, and at least 85% with the amino acid sequence shown in any of SEQ ID NOs: 3 to 10 The protein according to [12], which is selected from the group consisting of an amino acid sequence having identity and a polypeptide having immunoglobulin binding activity.
[14] The protein according to any one of [1] to [13], comprising 3 to 8 of the domains.
[15] The protein according to any one of [1] to [14], which is an affinity chromatography ligand.
[16] A polynucleotide encoding a protein according to any one of [1] to [15].
[17] A vector comprising the polynucleotide of [16].
[18] A vector transformant comprising the vector of [17].
[19] A carrier for affinity chromatography, which comprises a solid phase carrier and the protein according to any one of [1] to [15] bound to the solid phase carrier.
[20] [19] A method of isolating an antibody or a fragment thereof, using the carrier for affinity chromatography according to [20].

In the carrier for affinity chromatography of the present invention, the immunoglobulin binding ability of the ligand is improved by interposing a specific linker between the immunoglobulin binding domains in the ligand, and the immunoglobulin purification efficiency of the carrier for affinity chromatography is improved. It can be improved. In the present invention, the immunoglobulin dynamic binding capacity (DBC) and ligand utilization efficiency in a carrier for affinity chromatography can be obtained by modifying the linker that links the immunoglobulin binding domains in the ligand protein without mutating the immunoglobulin binding domains. Improve. The carrier for affinity chromatography of the present invention can be compared to a carrier containing a ligand protein using a conventional linker for immunoglobulin binding domain, for example, a linker consisting of a repeating unit of GGSGGS (Patent Document 1), an immunoglobulin DBC and a ligand Utilization efficiency is improved.

In the present specification, the identity of amino acid sequences and nucleotide sequences can be determined using the algorithm BLAST by Carlin and Artur (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, use the default parameters of each program. Specific procedures for these analysis methods are known (see www.ncbi.nlm.nih.gov).

In the present specification, "at least 85% identity" with respect to the amino acid sequence and nucleotide sequence means 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, more preferably Is 97% or more identity, more preferably 98% or more identity, still more preferably 99% or more identity.

In the present specification, the “corresponding position” on the amino acid sequence and nucleotide sequence refers to the conservation that the target sequence and the reference sequence (eg, the amino acid sequence shown in SEQ ID NO: 1) are present in each amino acid sequence or nucleotide sequence It can be determined by aligning the amino acid residues or nucleotides so as to give the greatest homology. Alignment can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al, 1994, Nucleic Acids Res., 22: 4673-4680) with default settings. Clustal W is, for example, the Japan Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) or the Japanese DNA databank (DDBJ [www. ddbj.nig.ac.jp/Welcome-j.html] can be used on the website.

In the present specification, amino acid residues are also described 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). Also herein, any acidic amino acid residue (eg aspartic acid or glutamic acid) is represented by X ^a and any basic amino acid residue (eg arginine, tryptophan, histidine or lysine) is represented by X ^b . Also in the present specification, the amino acid sequence of the peptide is described according to a conventional method such that the amino terminus (hereinafter referred to as N-terminus) is located on the left and the carboxyl terminus (hereinafter referred to as C-terminus) on the right.

In the present specification, protein A refers to protein A which is a cell wall component of Staphylococcus aureus (Staphylococcus aureus). As used herein, protein L refers to protein L, which is one of the proteins produced by Finegoldia magna.

As used herein, "immunoglobulin" (Ig) includes any class of immunoglobulin such as IgG, IgA, IgD, IgE, IgM, and their subclasses. The term "antibody" as used herein refers to an immunoglobulin or a fragment thereof containing an antigen recognition site, for example, an immunoglobulin of any class such as IgG, IgA, IgD, IgE, IgM and their subclasses, a fragment thereof, They may include variants thereof. Furthermore, the "antibody" in the present specification may be a chimeric antibody such as a humanized antibody, an antibody complex, and other modified immunoglobulins containing an antigen recognition site. In the present specification, "a fragment of an antibody" may be a fragment of an antibody containing an antigen recognition site or a fragment of an antibody not containing an antigen recognition site. Examples of antibody fragments that do not contain an antigen recognition site include proteins consisting of only the Fc region of immunoglobulins, Fc fusion proteins, and variants and modifications thereof.

As used herein, "a domain having affinity for immunoglobulin" or "immunoglobulin binding domain" refers to a functional unit of a polypeptide having immunoglobulin binding activity alone, which is contained in a protein. In the present specification, "having an affinity to an immunoglobulin" preferably refers to binding to a region other than the complementarity determining region (CDR) of an immunoglobulin molecule, and more preferably binding to at least an Fc fragment. To have. In addition, the domain which has the affinity to immunoglobulin may be only hereafter called a domain.

Preferred examples of the “domain having affinity to immunoglobulin” or “immunoglobulin binding domain” in the present specification include Fc binding protein, immunoglobulin binding domain of protein A, immunoglobulin binding domain of protein L, and immuno Those variants having globulin binding activity are mentioned. Among these, the immunoglobulin binding domain of protein A, the immunoglobulin binding domain of protein L, and variants thereof having immunoglobulin binding activity are mentioned as more preferable examples.

Examples of the immunoglobulin binding domain of protein A include protein A's A domain, B domain, C domain, D domain, E domain, and Z domain which is a modified domain of B domain, among which B More preferred are domain, Z domain, C domain and D domain. The A domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 1. The E domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 2. The B domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 3. The Z domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 4. The C domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 5. The D domain of protein A consists of the amino acid sequence shown in SEQ ID NO: 6.

Examples of the immunoglobulin binding domain of the protein L include the B1 domain, the B2 domain, the B3 domain, the B4 domain, and the B5 domain of protein L produced by the Finegoldia magna 312 strain; The C1 domain, the C2 domain, the C3 domain and the C4 domain of protein L produced by the magna 3316 strain can be mentioned, and among these, the C1 domain, the C2 domain, the C3 domain and the C4 domain are more preferable. The C1 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 7. The C2 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 8. The C3 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 9. The C4 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 10. The B1 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 11. The B2 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 12. The B3 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 13. The B4 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 14. The B5 domain of protein L consists of the amino acid sequence shown in SEQ ID NO: 15.

An example of a variant of the immunoglobulin binding domain of protein A comprises an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NOs: 1 to 6, and has immunoglobulin binding activity Polypeptides are included. Preferably, an example of a variant of the immunoglobulin binding domain of protein A comprises an amino acid sequence having at least 85% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, and immunoglobulin binding Included are polypeptides having activity. An example of a variant of the immunoglobulin binding domain of said protein L consists of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NO: 7 to 15, and has immunoglobulin binding activity Polypeptides are included. Preferably, as an example of a variant of the immunoglobulin binding domain of the protein L, it consists of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NOs: 7 to 10, and immunoglobulin binding Included are polypeptides having activity.

A variant of the above immunoglobulin binding domain is prepared by modifying the amino acid sequence of the domain by addition, deletion, substitution or deletion of amino acid residues, or chemical modification of amino acid residues. Can. Examples of means for addition, deletion, substitution or deletion of amino acid residues include known means such as site-specific mutation for a polynucleotide encoding the above-mentioned domain.

1. Proteins for Affinity Chromatography Ligands In one embodiment, the present invention provides proteins that function as affinity chromatography ligands. The protein of the present invention comprises two or more domains linked by a linker and having an affinity for immunoglobulin. In other words, the protein of the present invention comprises two or more domains linked by a linker, and the domains are domains having affinity for immunoglobulin. The linker linking the domains is preferably a peptide having a rod-like structure. The rod-like structure includes, for example, a needle-like, helical, rod-like structure, and preferably, the rod-like structure is helical or rod-like. In a preferred embodiment, the linker is a linker comprising at least one proline. In another preferred embodiment, the linker is a linker comprising a peptide having an acidic amino acid (X ^a ) and a basic amino acid (X ^b ).

The linker contained in the protein of the present invention is such that the N terminus is bound to any one of two or more domains having an affinity for the immunoglobulin constituting the protein of the present invention, and The C-terminus of the linker is linked to another domain that constitutes the protein of the invention. The position on the domain to which the linker binds is not particularly limited. Further, the number of the linkers bound to one domain is not particularly limited. Therefore, the protein of the present invention may have a structure in which two or more of the domains are connected in tandem with the linker, or a structure in which two or more of the domains are connected in parallel with the linker You may have.

More preferably, the linker contained in the protein of the present invention is such that the N terminus is bound to the C terminus of any one of two or more of the domains constituting the protein of the present invention, and The end is linked to the N-terminus of a domain different from the domain to which the N-terminus is linked. Preferably, the basic structure of the protein of the invention consists of repeats of this domain-linker unit. As a result, the protein of the present invention preferably has a structure in which two or more of the domains are linked in tandem via the linker. Alternatively, two or more of the linkers may be respectively linked to different positions of the C-terminal region of one domain, and different domains may be linked to the respective linkers (preferably, the N-terminus of the linker is Conjugated to the C-terminal region of one domain, the C-terminus of the linker being coupled to the N-terminus of another domain). The protein of the present invention so constructed may have a structure in which three or more of the domains are linked in parallel via the linker.

In a preferred embodiment of the invention, the linker comprised in the protein of the invention is a proline-containing linker. The proline-containing linker comprises at least one proline, preferably comprises two or more prolines, more preferably comprises four or more prolines, more preferably selected from the group consisting of Pro-Xaa and Xaa-Pro Containing four or more peptide units in total. Xaa contained in the proline-containing linker may be any amino acid including Pro, but preferably any amino acid other than Pro (ie, Ala, Arg, Asn, Asp, Cys, GIn, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val), more preferably Ala. More preferably, the proline-containing linker comprises a total of four or more peptide units selected from the group consisting of Pro-Ala and Ala-Pro. More preferably, the proline-containing linker consists of 4 or more Ala-Pro units. More preferably, the proline-containing linker consists of (Ala-Pro) n, where n is an integer, 4 or more, preferably 5 or more, more preferably 6 or more, and 25 or less. Preferably it is 15 or less, More preferably, it is 10 or less, More preferably, it is 9 or less. For example, n is an integer of 4 to 25, preferably 4 to 15, more preferably 5 to 15, still more preferably 4 to 10, still more preferably 5 to 10, still more preferably 5 to 9, more preferably 6 to 10 And more preferably 6 to 9.

Preferably, the proline-containing linker contained in the protein of the present invention is a polypeptide linker consisting of a polypeptide having a length of 8 amino acids or more, from the viewpoint of improving the avidity of the antibody to each domain. More preferably, the length of the proline-containing linker is 10 amino acids or more, and more preferably 12 amino acids or more. The upper limit of the length of the proline-containing linker is preferably 50 amino acids or less, more preferably 30 amino acids or less, still more preferably 22 amino acids or less, more preferably 20 amino acids or less from the viewpoint of stability of the expression amount of recombinant protein in E. coli It is an amino acid or less, more preferably 18 amino acids or less. Suitably, the proline-containing linker has a length of 8 to 50 amino acids, preferably 8 to 30 amino acids, more preferably 10 to 30 amino acids, still more preferably 10 to 22 amino acids, still more preferably 8 to 20 amino acids, more preferably Is 10 to 20 amino acids, more preferably 10 to 18 amino acids, more preferably 12 to 20 amino acids, and still more preferably 12 to 18 amino acids.

In another preferred embodiment of the present invention, the linker included in the protein of the present invention is a linker comprising a peptide having an acidic amino acid (X ^a ) and a basic amino acid (X ^b ) (herein, X ^a X ^b Also called a linker). The X ^a X ^b linker includes a peptide unit consisting of X ^a- (A) _i- X ^b or X ^b- (A) _i- X ^a , and the number of the peptide units contained in the X ^a X ^b linker Is at least one, preferably two or more; wherein X ^a is an acidic amino acid (eg aspartic acid or glutamic acid) and X ^b is a basic amino acid (eg arginine, tryptophan, (Histidine or lysine, preferably arginine or lysine), A is alanine, i is 2 or more, preferably an integer of 3 or 4. Preferably, the X ^a X ^b linker is Glu- (Ala) _i -Arg, Glu- (Ala) _i -Lys, Asp- (Ala) _i -Arg, Asp- (Ala) _i -Lys, Arg- ( _{Ala) i -Glu, Lys- (Ala} ) i -Glu, Arg- (Ala) i -Asp, and Lys- _(Ala) i peptide units (i selected from the group consisting -Asp 3 or 4 integer 2) or more in total. For example, the X ^a X ^b linker is selected from the group consisting of Glu- (Ala) _i- Arg, Glu- (Ala) _i- Lys, Asp- (Ala) _i- Arg, and Asp- (Ala) _i- Lys. The selected peptide units (i is an integer of 3 or 4) in total contain 2 or more. Further, for example, the ^X a ^{X b} _{linker, Arg- (Ala) i -Glu,} Lys- (Ala) i -Glu, Arg- (Ala) i -Asp, and Lys- _(Ala) group consisting of i -Asp It contains two or more peptide units (i is an integer of 3 or 4) selected in total. More preferably, the X ^a X ^b linker comprises 2 or more of Glu- (Ala) ₃ -Lys or 2 or more of Lys- (Ala) ₃ -Glu. More preferably, said X ^a X ^b linker consists of two or more Glu- (Ala) ₃ -Lys or consists of two or more Lys- (Ala) ₃ -Glu. More preferably, said X ^a X ^b linker consists of [Glu- (Ala) ₃ -Lys] _j , where j is an integer, 2 or more and 10 or less, preferably It is 8 or less, more preferably 6 or less, and still more preferably 4 or less. For example, j is an integer of 2 to 10, preferably 2 to 8, more preferably 2 to 6, and further preferably 2 to 4.

Preferably, the X ^a X ^b linker contained in the protein of the present invention is a polypeptide linker consisting of a polypeptide having a length of 10 amino acids or more, from the viewpoint of improving the avidity of the antibody to each domain. The upper limit of the length of the proline-containing linker is preferably 50 amino acids or less, more preferably 40 amino acids or less, still more preferably 30 amino acids or less, more preferably 20 amino acids or less from the viewpoint of stability of the expression amount of recombinant protein in E. coli etc. Less than amino acids. Suitably, the proline-containing linker has a length of 10 to 50 amino acids, more preferably 10 to 40 amino acids, more preferably 10 to 30 amino acids, and still more preferably 10 to 20 amino acids.

The number of the linkers contained in the protein of the present invention may be one or more, and varies depending on the number of domains. Typically, the number of linkers contained in the protein of the present invention is one less than the number of domains having affinity for immunoglobulin in the protein. When the protein of the present invention comprises two or more linkers, the amino acid sequence of each linker or the length thereof may be the same or different. Preferably, the amino acid sequences and lengths of the respective linkers are identical.

The protein of the present invention contains two or more, preferably 2 to 12, more preferably 3 to 8 domains having affinity for immunoglobulin. Each of two or more of the domains contained in the protein of the present invention may be the same domain or different domains, but is preferably the same domain.

In a preferred embodiment, each of the domains having affinity for the immunoglobulin is a protein A B domain, a Z domain, a C domain and a D domain, a protein L C1 domain, a C2 domain, a C3 domain and a C4 domain, and It is selected from the group consisting of those mutants.

In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NO: 3 to 10, and shown in any of SEQ ID NO: 3 to 10 It is selected from the group consisting of an amino acid sequence having at least 85% identity with the amino acid sequence, and a polypeptide having immunoglobulin binding activity.

In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3-10, with the proviso that any of SEQ ID NOs: 3-10 It is a polypeptide having Val at the position corresponding to position 1 of the amino acid sequence shown in In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NOs: 3-10. However, it is a polypeptide having Val at the position corresponding to position 1 of the amino acid sequence shown in any of SEQ ID NOs: 3 to 10. The reason why Val at position 1 is preferable is that the specific expression amount (weight-protien / weight-cell) by E. coli differs depending on the type of amino acid adjacent to the terminal methionine, and the specific expression amount of valine is larger than that of alanine (Proc Natl Acad Sci USA. 1989, 86 (21): 8247-8251. Fig 2).

In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3-10, with the proviso that any of SEQ ID NOs: 3-10 A polypeptide having Ala at the position corresponding to position 29 of the amino acid sequence shown in In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NOs: 3-10. However, it is a polypeptide having Ala at the position corresponding to position 29 of the amino acid sequence shown in any of SEQ ID NOs: 3 to 10. Position 29 Ala is preferred from the viewpoint of increasing the alkali resistance of the protein of the present invention (WO 2010/110288).

In a further preferred embodiment, each of the domains having affinity for the immunoglobulin is a Val1 / Ala29 variant of a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3-10, or SEQ ID NO: 3 10. A Val1 / Ala29 variant of a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence as set forth in any of ̃10.

In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3-10, with the proviso that any of SEQ ID NOs: 3-10 A polypeptide in which one amino acid is inserted between the position corresponding to position 3 and the position corresponding to position 4 of the amino acid sequence shown in In another preferred embodiment, each of the domains having affinity to the immunoglobulin is a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of SEQ ID NOs: 3-10. However, it is a polypeptide in which one amino acid is inserted between the position corresponding to position 3 and the position corresponding to position 4 of the amino acid sequence shown in any of SEQ ID NOs: 3 to 10. The amino acid to be inserted is selected from the group consisting of Ala, Arg, Asp, Gln, Glu, His, Met, Thr, Val, Phe, Leu, Ile, Pro, Trp and Tyr, preferably Leu. The insertion mutation is preferable from the viewpoint of improving the antibody binding capacity of the protein of the present invention (WO2016 / 152946). In a further preferred embodiment, each of the domains having affinity to the immunoglobulin is an N3K4_insL variant of a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3-10, or SEQ ID NOs: 3-10 And a N3K4_insL variant of a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence shown in any of the following.

In a further preferred embodiment, each of the domains having affinity to the immunoglobulin is a Val1 / Ala29 / N3K4_insL variant of a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3 to 10, or a sequence It is a Val1 / Ala29 / N3K4_insL variant of a polypeptide consisting of an amino acid sequence having at least 85% identity to the amino acid sequence represented by any one of Nos. 3 to 10. In a further preferred embodiment, each of the domains having affinity for said immunoglobulin is a polypeptide consisting of SEQ ID NO: 16.

Thus, in a preferred embodiment, the protein of the invention comprises a polypeptide represented by [(PL) mP]: P is a domain having affinity for the above mentioned immunoglobulins, preferably a sequence 16 is a polypeptide consisting of the amino acid sequence shown by the number 16, each P may be the same or different; L is, independently of each other, a proline-containing linker or an X ^a X ^b linker as described above Preferably, the proline-containing linker is a polypeptide consisting of the sequence shown in (Ala-Pro) n (where n is as described above); preferably, the X ^a X ^b linker is [Glu- (Ala ) ₃ [j is as described above] -Lys] _j is a polypeptide consisting of the sequence shown in; m is 1 or more, preferably 1 to 11, more preferably ~ Is 7. When m is 2 or more, a plurality of L may be the same or different.

In one embodiment, the protein of the present invention comprises the above linker and two or more domains having affinity for the above immunoglobulin, preferably a poly represented by the above [(PL) mP]. It consists of a peptide. In another embodiment, the protein of the present invention comprises the above linker and two or more domains having an affinity for the above immunoglobulin, preferably a polypeptide represented by the [(PL) mP]. In addition, it may further contain a linker for binding to a solid support. For example, each of one or more linkers for binding the protein of the present invention to a solid phase carrier may be bound at any position in the domain having affinity to the immunoglobulin. The linker for binding the protein of the present invention to a solid phase carrier may be bound to the C-terminus or N-terminus of the domain having affinity to the immunoglobulin, or an amino acid residue which is not a terminal residue May be combined with Preferably, a linker for linking the protein of the present invention to a solid phase carrier is linked to the C-terminus or N-terminus of the amino acid sequence of the polypeptide represented by [(PL) mP]. . Alternatively, each of one or more linkers for binding to the solid support may be bound to both ends of the amino acid sequence of the polypeptide represented by [(PL) mP]. Alternatively, the linker for binding to the solid phase carrier may be bound to an amino acid residue which is not a terminal residue. Examples of the linker for binding the protein of the present invention to a solid phase carrier include 1 or more and 20 or less Gly.

Preferred examples of the protein of the present invention include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 17, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 19 It can be mentioned. However, preferred examples of the protein of the present invention are not limited thereto.

2. Production of Protein for Affinity Chromatography Ligand The protein of the present invention can be produced by a method known in the art, such as a chemical synthesis method based on an amino acid sequence, a recombinant method and the like. For example, a protein of the invention can be produced by Frederick M. It can be manufactured using known genetic recombination techniques described in Ausbel et al., Current Protocols In Molecular Biology, and Sambrook et al., Molecular Cloning (Cold Spring Harbor Laboratory Press, 3rd edition, 2001). That is, the expression vector containing the polynucleotide encoding the protein of the present invention is transformed into a host such as E. coli, and the resulting recombinant is cultured in an appropriate liquid medium to obtain a cultured cell, The protein ligand of interest can be obtained in large amounts and economically. As a preferred expression vector, any of known vectors that can replicate in host cells can be used, for example, the plasmids described in US Pat. No. 5,151,350, and those edited by Sambrook et al. Examples include plasmids described in Molecular Cloning (Cold Spring Harbor Laboratory Press, 3rd edition, 2001) and the like. The host for transformation is not particularly limited, and known hosts used for expressing recombinant proteins such as bacteria such as E. coli, fungi, insect cells, mammalian cells and the like can be used. Depending on the host, any method known in the art may be used to transform the host by introducing the nucleic acid into the host, for example, Molecular Cloning (Cold Spring Harbor, edited by Sambrook et al.). The known methods described in Laboratory Press, 3rd edition, 2001) can be used. Methods for culturing transformed recombinants (such as bacteria) and recovering the expressed protein are well known to those skilled in the art.

Therefore, the present invention also provides a polynucleotide (such as DNA) encoding the above-mentioned protein of the present invention, a vector containing it, and a transformant containing them.

3. Carrier for Affinity Chromatography A carrier for affinity chromatography can be produced by immobilizing the protein for affinity chromatography ligands of the present invention on a solid phase carrier.

The form of the solid phase carrier contained in the carrier for affinity chromatography of the present invention may be in the form of particles, and such particles may be porous or non-porous. The particulate carrier can be used as a packed bed or in suspension form. Suspension forms include what are known as expanded beds and pure suspensions, in which the particles can move freely. In the case of monoliths, packed beds and fluidized beds, the separation procedure generally follows conventional chromatographic methods with concentration gradients. In the case of pure suspensions, the batch method is used. Preferably, the carrier is a filler. Alternatively, the carrier may be in the form of a chip, capillary or filter.

In one embodiment, the solid support is preferably 20 to 200 μm, more preferably 20 to 100 μm, more preferably 30 to 80 μm when the carrier is a synthetic polymer, more preferably 50 to 200 μm when the carrier is a polysaccharide. More preferably, it has a particle size of 60 to 150 μm. If the particle size is less than 20 μm, the column pressure will be high at high flow rates, which is not practical. When the particle size exceeds 200 μm, the amount of immunoglobulin bound to the carrier for affinity chromatography (binding capacity) may be poor. The “particle size” in the present specification is a volume average particle size obtained by a laser diffraction scattering type particle size distribution measuring apparatus.

In one embodiment, the solid support is preferably porous and has a specific surface area of 50 to 150 m ² / g, more preferably 80 to 130 m ² / g. Here, if the specific surface area is less than 50 m ² / g, the binding capacity may be inferior, while if it exceeds 150 m ² / g, the strength of the support is inferior and the support is broken under high flow rate, Column pressure may increase. The “specific surface area” in the present specification is a value obtained by dividing the surface area of pores having a pore diameter of 10 to 5000 nm obtained by a mercury porosimeter by the dry weight of particles.

In one embodiment, the solid support is preferably 100 to 1400 nm, more preferably 100 to 400 nm, more preferably 200 to 300 nm when the support is a synthetic polymer, more preferably 500 to 500 nm when the support is a polysaccharide. It has a volume average pore size of 1400 nm, more preferably 800 to 1200 nm. Here, if the volume average pore diameter is less than 100 nm, the decrease in binding capacity under high flow rate may be remarkable, while if it exceeds 1400 nm, the binding capacity may decrease regardless of the flow rate. The “volume-average pore diameter” in the present specification is the volume-average pore diameter of pores with a pore diameter of 10 to 5000 nm obtained by a mercury porosimeter.

When the solid phase support satisfies the particle diameter, specific surface area, and pore diameter distribution in the above range, the gaps between the particles serving as the flow path of the solution to be purified and the relatively large pore diameter in the particles and the binding of the molecule to be purified The surface area balance is optimized to keep the binding capacity at high flow rates at high levels.

The material of the solid phase support is, for example, a polymer having a hydrophilic surface, and for example, a hydroxy group (-OH), a carboxy group (-COOH) on the outer surface (and also on the inner surface if present) , An aminocarbonyl group (-CONH ₂ or N-substituted type), an amino group (-NH ₂ or substituted type), or a polymer having an oligo or polyethyleneoxy group. In one embodiment, the polymer may be a synthetic polymer such as polymethacrylate, polyacrylamide, polystyrene, polyvinyl alcohol and the like, and preferably, it is preferably co-crosslinked with a polyfunctional monomer such as polyfunctional (meth) acrylate and divinylbenzene. It is a synthetic polymer such as a polymer. Such synthetic polymers are readily prepared by known methods (see, for example, the methods described in J. MATER. CHEM 1991, 1 (3), 371-374). Alternatively, commercially available products such as Toyopearl (Tosoh Corporation) are also used. In another embodiment, the polymer is a polysaccharide such as dextran, starch, cellulose, pullulan, agarose and the like. Such polysaccharides are easily produced by known methods (see, for example, the method described in Patent No. 4081143). Alternatively, commercially available products such as Sepharose (GE Healthcare Biosciences) are also used. In other embodiments, it may be an inorganic support such as silica or zirconium oxide.

In one embodiment, as a specific example of the porous particles used as the solid phase carrier, for example, 20 to 50% by mass of a crosslinkable vinyl monomer and 3 to 80% by mass of an epoxy group-containing vinyl monomer Body, containing 20 to 80% by mass of a copolymer with a diol group-containing vinyl monomer, having a particle size of 20 to 80 μm, a specific surface area of 50 to 150 m ² / g, and a volume average pore diameter Porous organic polymer particles having a size of 100 to 400 nm can be mentioned.

The penetration volume (pore volume) of pores with a pore diameter of 10 to 5000 nm when the solid support is measured with a mercury porosimeter is preferably 1.3 to 7.0 mL / g, and the carrier is a synthetic polymer. In the case of a polysaccharide, the carrier is more preferably 1.3 to 2.5 mL / g, and more preferably 3.0 to 6.0 mL / g.

The method for attaching a ligand (ie, the protein of the present invention) to the solid phase carrier can be carried out using a general method of immobilizing a protein on a carrier. For example, using a carrier having a carboxy group, the carboxy group is activated with N-hydroxysuccinimide and reacted with the amino group of the ligand; using a carrier having an amino group or a carboxy group, dehydration of a water-soluble carbodiimide or the like Method of reacting with the carboxy group or amino group of the ligand to form an amide bond in the presence of a condensing agent; using a carrier having a hydroxyl group, activating with a cyan halide such as cyanogen bromide and reacting with the amino group of the ligand A method of tosylation or tresylation of the hydroxyl group of the carrier to react with the amino group of the ligand; a method of introducing an epoxy group into the carrier by bisepoxide, epichlorohydrin etc. and reacting with the amino group, hydroxyl group or thiol group of the ligand; Using a carrier having an epoxy group, the amino group of the ligand, hydroxyl group or thio A method of reacting a group, and the like. Among the above, from the viewpoint of stability in an aqueous solution in which the reaction is carried out, a method of binding a ligand via an epoxy group is desirable.

The alcoholic hydroxyl group, which is a ring-opened epoxy group formed by ring-opening of an epoxy group, hydrophilizes the surface of the carrier to prevent non-specific adsorption of proteins etc. and improve the toughness of the carrier in water, under high flow rate. It serves to prevent the destruction of the carrier. Therefore, when the residual epoxy group not bound to the ligand is present in the carrier after the ligand is immobilized, it is preferable to open the residual epoxy group. As a ring-opening method of the epoxy group in a support | carrier, the method of stirring this support | carrier at heating or room temperature with an acid or an alkali in water solvent can be mentioned, for example. Alternatively, the epoxy group may be ring-opened with a blocking agent having a mercapto group such as mercaptoethanol and thioglycerol, or a blocking agent having an amino group such as monoethanolamine. A more preferable ring-opened epoxy group is a ring-opened epoxy group obtained by ring-opening an epoxy group contained in a carrier with thioglycerol. Thioglycerol is less toxic than mercaptoethanol etc. as a raw material, and the epoxy ring-opened group to which thioglycerol is added has lower non-specific adsorption than the ring-opened group by the blocking agent having an amino group, and dynamic binding It has the advantage that the amount is high.

Preferably, the solid support and the protein of the present invention are linked via a linker for binding the protein of the present invention described above to the solid support. Preferably, the linker is contained in the protein of the present invention as described above, and the linker moiety reacts with the solid phase carrier to bind the solid phase carrier and the protein of the present invention. Alternatively, the protein of the present invention can be bound to the linker previously bound to the solid support.

The carrier for affinity chromatography of the present invention has a ligand with high immunoglobulin binding ability, and thus has high immunoglobulin dynamic binding capacity (DBC) and high ligand utilization efficiency.

4. Methods of Isolating Antibodies or Fragments Thereof A method of isolating an antibody or fragment thereof according to one embodiment of the present invention is described. The method for isolating the antibody or fragment thereof according to the present embodiment is carried out by passing the sample containing the antibody or fragment thereof into the carrier for affinity chromatography on which the protein for affinity chromatography ligand of the present invention is immobilized. A step of adsorbing the antibody or fragment thereof to the carrier (first step), and a step of eluting the antibody or fragment thereof from the carrier (second step) are included.

In the first step, the sample containing the antibody or its fragment is flowed in a column or the like packed with the carrier for affinity chromatography of the present invention under the condition that the antibody or its fragment is adsorbed to the ligand (the protein of the present invention) . In this first step, most of the substances other than the antibody or fragment thereof in the sample pass through the column without being adsorbed to the ligand. After this, the carrier may be washed with a neutral buffer containing a salt such as NaCl, if necessary, in order to remove some of the substances weakly retained by the ligand.

In the second step, an appropriate buffer of pH 2-5 is applied to elute the antibody or fragment thereof adsorbed to the ligand. By collecting this eluate, the antibody or fragment thereof can be isolated from the sample.

In one embodiment of the method for isolating an antibody or fragment thereof of the present invention, the antibody or fragment thereof to be isolated may be an antibody or fragment thereof, or a medicament comprising them. Therefore, in one embodiment, the present invention provides a method for producing an antibody drug using the carrier for affinity chromatography of the present invention. The procedure of the method is basically the same as the procedure of the method for isolating an antibody or a fragment thereof described above except that a sample containing an antibody drug of interest is used.

Hereinafter, the present invention will be described in more detail by way of examples. In addition, the following description generally shows the embodiments of the present invention, and the present invention is not limited by the description without any particular reason.

Reference Example 1 Synthesis of Porous Particles (1) 3.58 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray) is added to 360 g of pure water, heated and stirred to dissolve polyvinyl alcohol, and after cooling, sodium dodecyl sulfate 0.36 g (manufactured by Wako Pure Chemical Industries, Ltd.), 0.36 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.18 g of sodium nitrite (manufactured by Wako Pure Chemical Industries) were added and stirred to prepare an aqueous solution S. .
(2) A monomer composition consisting of 12.00 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon) and 1.33 g of divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.) is dissolved in 24.43 g of diisobutyl ketone (manufactured by Mitsui Chemicals) The monomer solution was prepared.
(3) The entire amount of the aqueous solution S was charged into a separable flask, and a thermometer, a stirring blade and a cooling pipe were attached, and the solution S was set in a hot water bath, and stirring was started under a nitrogen atmosphere. The whole amount of the monomer solution was charged into the separable flask and heated by a hot water bath. When the internal temperature reached 85 ° C., 0.53 g of 2,2′-azoisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) was added.
(4) The resulting reaction solution was stirred for 3 hours while maintaining the temperature at 86 ° C. Then, after cooling the reaction solution, 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 so that the concentration of the particles was 10% by mass, to obtain a porous particle (PB) dispersion.

Comparative Example 1 Preparation of Recombinant Ligand Protein: IgGBP0 A plasmid encoding a recombinant ligand protein IgGBP0 consisting of the amino acid sequence shown in SEQ ID NO: 20, containing four repeats of the amino acid sequence shown in SEQ ID NO: 16 was prepared. E. coli competent cell BL21 (DE3) (manufactured by NEW ENGLAND BIOLABS) was transformed using this plasmid. The resulting recombinants are incubated at 37 ° C. until the absorbance (OD600) reaches about 10, and then IPTG (manufactured by Wako Pure Chemical Industries, Ltd.) is added to a final concentration of 1 mM for an additional 5 hours The recombinant protein was expressed by incubation at 37 ° C. After protein expression, cells were harvested by centrifugation and dispersed in Tris buffer, pH 8.5. To this, Triton X-100 was added to destroy E. coli. The recombinant protein was purified from the resulting disrupted cell suspension by cation exchange chromatography (BioProS 75, YMC) and anion exchange chromatography (BioProQ 75, YMC). The purified protein was concentrated and desalted by Tangential Flow Filteration against 10 mM citrate buffer. The theoretical molecular weight [Da] of the obtained recombinant ligand protein was determined using ExPASy ([web.expasy.org/protparam/]). Also, the purity and molecular weight of the recombinant ligand protein were measured using LC-MS (Waters). The purity (LC purity) was determined as a percentage of the peak area of IgGBP0 to the total area of all peaks detected on the chromatogram of LC-MS.

Examples 1 to 3 Preparation of recombinant ligand protein: IgGBP1 to IgGBP3 A plasmid encoding a recombinant ligand protein IgGBP1 consisting of the amino acid sequence shown in SEQ ID NO: 17, a recombinant ligand protein IgGBP2 consisting of the amino acid sequence shown in SEQ ID NO: 18 And a plasmid encoding a recombinant ligand protein IgGBP3 consisting of the amino acid sequence shown in SEQ ID NO: 19. Using these plasmids, a recombinant ligand protein was prepared in the same manner as in Comparative Example 1. IgGBPs 1, 2 and 3 contain the amino acid sequence shown by SEQ ID NO: 16 linked by an inter-domain linker shown in Table 1, respectively. The theoretical molecular weight, LC purity and measured molecular weight of the obtained recombinant ligand protein were measured in the same manner as in Comparative Example 1.

Comparative Example 2 Preparation of Recombinant Ligand Protein: IgGBP4 A plasmid encoding a recombinant ligand protein IgGBP4 consisting of the amino acid sequence shown in SEQ ID NO: 21 was prepared. Using this plasmid, a recombinant ligand protein was prepared in the same manner as in Comparative Example 1. IgGBP4 comprises the amino acid sequence shown by SEQ ID NO: 16 linked by an inter-domain linker (Patent Document 1) consisting of repeating units of GGSGGS. The theoretical molecular weight, LC purity and measured molecular weight of the obtained recombinant ligand protein were measured in the same manner as in Comparative Example 1.

The LC purity and molecular weight of the recombinant ligand proteins of Comparative Examples 1-2 and Examples 1-3 are shown in Table 1.

Test Example 1
(1) Preparation of Ligand Protein-Immobilized Porous Particles With respect to the porous particles PB obtained in Reference Example 1, 1.2 M sodium sulfate / 0. 2 so that the amount of ligand protein per 1 g of particles is 0.15 g. The ligand protein of Example 1-3 or Comparative Example 1-2 and PB are mixed in a solution containing 1 M sodium carbonate buffer (pH 8.8), shaken for 5 hours at 25 ° C., ligand protein-immobilized porous particles ( IgGBP0 / PB, IgGBP1 / PB, IgGBP2 / PB, IgGBP3 / PB, and IgGBP4 / BP) were obtained. The epoxy groups remaining on these particles were blocked with thioglycerol. The particles were then washed with 0.5 M NaOH and 0.1 M citrate buffer (pH 3.2) and finally suspended in PBS.

(2) Measurement of Ligand Binding Amount The amount of ligand protein bound to particles was measured using a BCA Assay kit (PIERCE) for a suspension of 150 μL containing 1 mg of IgGBP0 / PB. The amount of ligand protein bound to the particles in IgGBP1 / PB to IgGBP4 / PB was measured in the same manner.

(3) Measurement of IgG Dynamic Binding Capacity (DBC) IgGBP0 / BP was packed in a column with an inner diameter of 0.5 cm to a bed height of 20 cm. After equilibrating the column with 20 mM phosphate buffer (pH 7.5), flow 20 mM phosphate buffer (pH 7.5) containing human polyclonal IgG (5 mg / mL) at a linear flow rate of 300 cm / hour and elute with absorbance monitor Dynamic binding capacity (DBC) was determined from the amount of human polyclonal IgG adsorbed and the carrier volume when the concentration of human polyclonal IgG in the solution was 10% breakthrough (breakthrough). The DBC of IgGBP1 / PB to IgGBP4 / PB was determined in the same manner. Furthermore, for each particle, DBC was divided by the amount of ligand binding, and the relative value (%) of the obtained value to the value of Comparative Example 1 was determined to calculate the ligand utilization efficiency.

As shown in Table 2, the particles immobilized with any of the ligand proteins IgGBP1 to 3 which are ligand proteins respectively containing the inter-domain linkers of Examples 1 to 3 are the ligand proteins which do not contain the linker of Comparative Example 1 are IgGBP0. DBC and ligand utilization efficiency was improved as compared to immobilized particles. Furthermore, it was found from the comparison of Examples 1 and 2 that the ligand utilization efficiency tends to further increase as the length of the inter-domain linker increases.

In addition, particles on which any one of IgGBPs 1 to 3 is immobilized are compared to particles on which IgGBP 4 (comparative example 2), which is a ligand protein containing a linker (Patent Document 1) consisting of repeating units of GGSGGS, is immobilized. DBC and ligand utilization efficiency was improved.

The explanation according to the embodiment of the present invention is as described above. However, the present invention is not limited to the embodiments described above, and various modifications of the present invention are possible. Furthermore, the invention includes configurations substantially the same as the configurations of the embodiments described above (for example, configurations having the same function, method and result, or configurations having the same purpose and result). The present invention also includes configurations in which nonessential parts of the configurations of the embodiments described above are replaced. The present invention also includes configurations that can achieve the same effects or the same objects as the configurations of the embodiments described above. The present invention also includes a configuration in which a known technique is added to the configuration of the embodiment described above.

Claims (20)

1. A protein comprising two or more domains linked by a linker, wherein the domains have an affinity for immunoglobulin and the linker is a peptide having a rod-like structure.
2. A protein comprising two or more domains linked by a linker, wherein
   The domain has an affinity for immunoglobulins and
   The linker, a linker comprising a polypeptide comprising at least one proline (a), and _{^{X a - (A) i -X}} b or ^X b - at least one _(A) consisting of i -X ^a peptide units Containing at least one of the following linkers (b) (wherein X ^a is an acidic amino acid, X ^b is a basic amino acid, A is alanine and i is an integer of 3 or 4): ,
   protein.
3. The protein according to claim 2, wherein the linker (a) consists of a polypeptide having a length of 8 amino acids or more.
4. The protein according to claim 3, wherein the linker (a) comprises 4 or more in total of peptide units selected from the group consisting of Pro-Xaa and Xaa-Pro.
5. 5. The protein of claim 4, wherein Xaa is an amino acid other than Pro.
6. 5. The protein of claim 4, wherein Xaa is Ala.
7. The protein according to claim 4, wherein the linker (a) consists of 4 or more Ala-Pro units.
8. The protein according to claim 2, wherein the linker (b) consists of a polypeptide having a length of 10 amino acids or more.
9. The linker (b) is Glu- (Ala) _i- Arg, Glu- (Ala) _i- Lys, Asp- (Ala) _i- Arg, Asp- (Ala) _i- Lys, Arg- (Ala) _{i- Glu, Lys- (Ala) i -Glu} , Arg- (Ala) i -Asp, and Lys- _(Ala) i comprises two or more in total peptide units selected from the group consisting -Asp, claim 2, wherein Protein.
10. The protein according to claim 9, wherein the peptide unit is Glu- (Ala) _3- Lys or Lys- (Ala) _3- Glu.
11. 11. The method according to any one of claims 1 to 10, wherein the N terminus of the linker is attached to the C terminus of any one of the two or more domains, and the C terminus of the linker is attached to the N terminus of another domain. Described protein.
12. The protein according to any one of claims 1 to 11, wherein each of the two or more domains is an immunoglobulin binding domain of protein A or protein L or a variant thereof.
13. Each of the two or more domains has at least 85% identity to a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 3 to 10, and the amino acid sequence shown in any of SEQ ID NOs: 3 to 10 The protein according to claim 12, which is selected from the group consisting of an amino acid sequence having and a polypeptide having an immunoglobulin binding activity.
14. The protein according to any one of claims 1 to 13, comprising 3 to 8 of the domains.
15. The protein according to any one of claims 1 to 14, which is an affinity chromatography ligand.
16. A polynucleotide encoding a protein according to any one of claims 1-15.
17. A vector comprising the polynucleotide according to claim 16.
18. A vector transformant comprising the vector according to claim 17.
19. A carrier for affinity chromatography, comprising a solid phase carrier and the protein according to any one of claims 1 to 15 bound to the solid phase carrier.
20. A method of isolating an antibody or a fragment thereof using the affinity chromatography carrier according to claim 19.