IgG-binding peptides and methods of using the same to detect and purify igg

Abstract

The present invention provides a peptide having specificity or selectivity of binding to human IgG. The peptide is characterized by comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by Formula I, and is capable of binding to human IgG: (X 1-3 )-C-(X2)-H-R-G-(Xaa1)-L-V-W-C-(X 1-3 ), wherein X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, R is an arginine residue, G is a glycine residue, Xaa1 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue.

Description

Technical Field

[0001] This invention relates to a human IgG-binding peptide obtained from a random peptide library and a method for detecting and purifying IgG using this peptide. Background Technology

[0002] Currently, antibody drugs are receiving significant attention as the most reliable molecularly targeted therapies, rapidly expanding into new drug areas. Most antibody drugs currently under development or in use utilize antibodies belonging to the immunoglobulin G (IgG) class.

[0003] Traditionally, IgG antibody purification has utilized proteins A or G derived from Staphylococcus aureus (Non-Patent Literature 1, 2). Because these proteins also bind to mouse and rabbit IgG, they are primarily used for IgG purification at the reagent level. However, in recent years, antibody drugs centered on human IgG1 have begun to be used in the pharmaceutical field, and their importance in industrial and pharmaceutical applications is increasing. In particular, protein A columns play a central role in antibody drug purification, and many antibody drug manufacturers have introduced purification systems centered around this column.

[0004] However, several problems have been identified with protein A columns. One of these is the issue of protein A contamination in purified antibodies. Protein A is a bacterial protein with high immunogenicity after human administration, and there are also concerns about endotoxin contamination. As an affinity ligand used for such drug purification, protein A, as the ligand, requires a high degree of purification to avoid the introduction of unsuitable substances. This is a major reason for the increased cost of protein A columns used for drug purification.

[0005] To address these issues, new purification systems for IgG antibodies have been developed. For example, reports have described non-peptide affinity ligands designed based on protein A mimic peptides (Non-Patent Literature 3, 4) or on the X-ray crystallographic structures of the Fc of protein A and IgG antibodies (Non-Patent Literature 5). However, their application is limited due to issues with their binding affinity and specificity.

[0006] In addition, there have been many studies exploring new IgG-binding peptides using phage libraries or synthetic peptide libraries (Patent Documents 1-3).

[0007] As mentioned above, although research has been conducted on the purification of IgG antibodies using novel peptides and low molecular weight IgG, there is no new purification system that can be used on an industrial scale to replace protein A and G columns. Therefore, a new method for purifying IgG antibodies is still being sought in this field.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: WO01 / 045746

[0011] Patent Document 2: WO02 / 086070

[0012] Patent Document 3: WO02 / 38592

[0013] Non-patent literature

[0014] Non-patent literature 1: Ey, PL, Prowse, SJ, and Jenkin, CR (1978) Immunochemistry 15(7), 429-436

[0015] Non-patent literature 2: Akerstrom, B., Brodin, T., Reis, K., and Bjorck, L. (1985) J Immunol 135(4), 2589-2592

[0016] Non-patent document 3: Fassina, G., Verdoliva, A., Odierna, MR, Ruvo, M., and Cassini, G. (1996) J Mol Recognit9 (5-6), 564-569

[0017] Non-patent literature 4: Fassina, G., Palombo, G., Verdoliva, A., and Ruvo, M. (2002) Affinity Purification of Immunoglobulins Using Protein A Mimetic (PAM) In: Walker, JM (ed). The Protein Protocols Handbook, Second Edition, Humana Press Inc., Totowa, NJ

[0018] Non-patent literature 5: Li, R., Dowd, V., Stewart, DJ, Burton, SJ, and Lowe, CR (1998) Nature biotechnology 16(2), 190-195 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] The purpose of this invention is to provide a peptide that specifically or selectively binds to human IgG.

[0021] Another object of the present invention is to provide a method for purifying or analyzing (detecting or quantifying) human IgG using the peptide.

[0022] Methods for solving problems

[0023] Human IgG is mainly found in the blood and plays an important role in the body's defense and homeostasis by eliminating foreign substances and through antibody-dependent cytotoxicity. In particular, due to these properties, IgG has been used as an antibody drug in recent years as a treatment primarily for cancer and autoimmune diseases such as rheumatic diseases. Based on the aforementioned important role of IgG antibodies as pharmaceuticals, this invention provides a peptide that can specifically or selectively bind to human IgG (especially IgG1). Therefore, it is believed that this is useful for solving the existing problems described in the background art and establishing methods for the purification and analysis of IgG that can be used as a pharmaceutical.

[0024] The present invention can be summarized as having the following characteristics.

[0025] [1] A peptide, characterized in that it contains an amino acid sequence of 13 to 17 amino acid residues as shown in Formula I, and is capable of binding to human IgG:

[0026] (X 1-3 )-C-(X2)-HRG-(Xaa1)-LVWC-(X 1-3 (I)

[0027] In the formula, X represents any amino acid residue other than cysteine.

[0028] C is a cysteine ​​residue.

[0029] H stands for histidine residue.

[0030] R represents an arginine residue.

[0031] G stands for a glycine residue.

[0032] Xaa1 is a glutamic acid residue or an asparagine residue.

[0033] L represents a leucine residue.

[0034] V represents a valine residue.

[0035] And W is a tryptophan residue.

[0036] [2] The peptide of [1] is characterized by containing an amino acid sequence of 13 to 17 amino acid residues as shown in Formula II below, and is capable of binding to human IgG:

[0037] (X 1-3 )-C-(Xaa2)-(Xaa3)-HRG-(Xaa1)-LVWC-(X 1-3 (II)

[0038] In the formula, X represents any amino acid residue other than cysteine.

[0039] C is a cysteine ​​residue.

[0040] H stands for histidine residue.

[0041] R represents an arginine residue.

[0042] G stands for a glycine residue.

[0043] Xaa1 is a glutamic acid residue or an asparagine residue.

[0044] L represents a leucine residue.

[0045] V represents a valine residue.

[0046] W represents a tryptophan residue.

[0047] Xaa2 is an alanine residue, a serine residue, or a threonine residue, and

[0048] Xaa3 is a tyrosine residue or a tryptophan residue.

[0049] [3] The peptide as in [1] or [2] is characterized by containing an amino acid sequence of 13 to 17 amino acid residues as shown in Formula III below, and is capable of binding to human IgG:

[0050] (X 1-3 )-CAYHRGELVWC-(X 1-3 (III)

[0051] In the formula, X represents any amino acid residue other than cysteine.

[0052] C is a cysteine ​​residue.

[0053] A is an alanine residue.

[0054] Y represents a tyrosine residue.

[0055] H stands for histidine residue.

[0056] R represents an arginine residue.

[0057] G stands for a glycine residue.

[0058] E represents a glutamic acid residue.

[0059] L represents a leucine residue.

[0060] V represents a valine residue.

[0061] And W is a tryptophan residue.

[0062] [4] For any of the peptides in [1] to [3], where, if it is set to have 17 amino acid residues, the amino acid residues from the N-terminus 1 to 3 and 15 to 17 are:

[0063] The first amino acid residue is either S, G, F, or absent;

[0064] The second amino acid residue is either D, G, A, S, P, or absent;

[0065] The third amino acid residue = S, D, T, N, E or R;

[0066] The 15th amino acid residue = S, T or D;

[0067] The 16th amino acid residue = H, G, Y, T, N, D, F or none;

[0068] The 17th amino acid residue is either Y, F, H, M, or absent.

[0069] [5] The peptide as described in [4] is composed of any of the following amino acid sequences 1) to 12).

[0070] 1)DCAYHRGELVWCT (SEQ ID NO: 55)

[0071] 2)GPDCAYHRGELVWCTFH (SEQ ID NO: 56)

[0072] 3)RCAYHRGELVWCS (SEQ ID NO: 57)

[0073] 4)GPRCAYHRGELVWCSFH (SEQ ID NO: 58)

[0074] 5)SPDCAYHRGELVWCTFH (SEQ ID NO: 100)

[0075] 6) GDDCAYHRGELVWCTFH (SEQ ID NO: 101)

[0076] 7) GPSCAYHRGELVWCTFH (SEQ ID NO: 102)

[0077] 8)GPDCAYHRGELVWCSFH (SEQ ID NO: 103)

[0078] 9)GPDCAYHRGELVWCTHH (SEQ ID NO: 104)

[0079] 10)GPDCAYHRGELVWCTFY (SEQ ID NO: 105)

[0080] 11)SPDCAYHRGELVWCTFY (SEQ ID NO: 106)

[0081] 12)SDDCAYHRGELVWCTFY (SEQ ID NO: 107)

[0082] [6] The peptide as in [1] or [2] is characterized by containing an amino acid sequence of 13 amino acid residues as shown in Formula IV below, and is capable of binding to human IgG:

[0083] DC-(Xaa2)-(Xaa3)-HRG-(Xaa1)-LVWCT (IV)

[0084] In the formula,

[0085] D stands for aspartic acid residue.

[0086] C is a cysteine ​​residue.

[0087] H stands for histidine residue.

[0088] R represents an arginine residue.

[0089] G stands for a glycine residue.

[0090] Xaa1 is a glutamic acid residue or an asparagine residue.

[0091] L represents a leucine residue.

[0092] V represents a valine residue.

[0093] W represents a tryptophan residue.

[0094] T stands for a threonine residue.

[0095] Xaa2 is an alanine residue or a threonine residue.

[0096] Furthermore, Xaa3 is either a tyrosine residue or a tryptophan residue.

[0097] [7] As in [6], the peptide is composed of any of the amino acid sequences 1) to 4) below.

[0098] 1)DCTYHRGNLVWCT (SEQ ID NO: 47)

[0099] 2)DCAYHRGNLVWCT (SEQ ID NO: 48)

[0100] 3)DCTYHRGELVWCT (SEQ ID NO: 50)

[0101] 4) DCAWHRGELVWCT (SEQ ID NO: 53).

[0102] [8] A peptide characterized in that it contains an amino acid sequence of 13 amino acid residues as shown in Formula V, and is capable of binding to human IgG:

[0103] DC-(Xaa1)-(Xaa2)-(Xaa3)-(Xaa4)-G-(Xaa5)-L-(Xaa6)-WCT (V)

[0104] In the formula,

[0105] D stands for aspartic acid residue.

[0106] C is a cysteine ​​residue.

[0107] G stands for a glycine residue.

[0108] L represents a leucine residue.

[0109] W represents a tryptophan residue.

[0110] T stands for a threonine residue.

[0111] Xaa1 is an alanine residue, a serine residue, or a threonine residue.

[0112] Xaa2 is a tryptophan residue or a tyrosine residue.

[0113] Xaa3 is a histidine residue, arginine residue, serine residue, or threonine residue.

[0114] Xaa4 is an asparagine residue or an arginine residue.

[0115] Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue, and

[0116] Xaa6 is an isoleucine residue or a valine residue.

[0117] [9] A peptide as described in [8], wherein the peptide is composed of any of the amino acid sequences 1) to 12) below.

[0118] 1)DCTYTNGNLVWCT (SEQ ID NO: 29)

[0119] 2)DCAYTNGNLVWCT (SEQ ID NO: 31)

[0120] 3) DCSYTNGNLVWCT (SEQ ID NO: 32)

[0121] 4)DCTWTNGNLVWCT (SEQ ID NO: 34)

[0122] 5)DCTYHNGNLVWCT (SEQ ID NO: 35)

[0123] 6)DCTYRNGNLVWCT (SEQ ID NO: 36)

[0124] 7)DCTYSNGNLVWCT (SEQ ID NO: 37)

[0125] 8)DCTYTRGNLVWCT (SEQ ID NO: 39)

[0126] 9)DCTYTNGELVWCT (SEQ ID NO: 40)

[0127] 10)DCTYTNGRLVWCT (SEQ ID NO: 41)

[0128] 11)DCTYTNGDLVWCT (SEQ ID NO: 42)

[0129] 12)DCTYTNGNLIWCT (SEQ ID NO: 45)

[0130]

[10] A peptide as in any of [1] to [9], wherein the peptide forms a disulfide bond between two cysteine ​​(C) residues.

[0131]

[11] Any peptide as in [1] to

[10] , wherein the peptide is linked to a label.

[0132]

[12] A fusion protein consisting of a peptide of any one of [1] to

[11] and a linked protein.

[0133]

[13] An immobilized peptide, which is formed by combining a peptide from any one of [1] to

[11] with an immobilized peptide.

[0134]

[14] A nucleic acid that encodes a peptide of any one of [1] to

[11] .

[0135]

[15] A method for purifying IgG, the method comprising binding IgG with a peptide of any one of [1] to

[11] or an immobilized peptide of

[13] , and releasing the bound IgG to recover the IgG.

[0136]

[16] A method for detecting IgG, the method comprising detecting the bound IgG by binding IgG in a sample to a peptide of any one of [1] to

[11] or an immobilized peptide of

[13] .

[0137]

[17] A kit for the analysis or purification of human IgG, the kit containing at least one of the peptides of any one of [1] to

[11] or an immobilized peptide of

[13] .

[0138]

[18] An IgG separation column containing an immobilized peptide of

[13] .

[0139]

[19] The peptide of [1] is characterized by containing an amino acid sequence of 13 to 17 amino acid residues as shown in Formula I' below, and is capable of binding to human IgG:

[0140] (X 1-3 )-C-(X1)-YHRGNLVWC-(X 1-3 ) (I')

[0141] In the formula, X represents any amino acid residue other than cysteine.

[0142] C is a cysteine ​​residue.

[0143] Y represents a tyrosine residue.

[0144] H stands for histidine residue.

[0145] R represents an arginine residue.

[0146] G stands for a glycine residue.

[0147] N represents an asparagine residue.

[0148] L represents a leucine residue.

[0149] V represents a valine residue.

[0150] And W is a tryptophan residue.

[0151]

[20] The peptide of [1] is characterized by containing an amino acid sequence of 13 to 17 amino acid residues as shown in Formula I'', and is capable of binding to human IgG:

[0152] (X 1-3 )-CA-(X1)-HRGELVWC-(X 1-3 ) (I'')

[0153] In the formula, X represents any amino acid residue other than cysteine.

[0154] C is a cysteine ​​residue.

[0155] A is an alanine residue.

[0156] H stands for histidine residue.

[0157] R represents an arginine residue.

[0158] G stands for a glycine residue.

[0159] E represents a glutamic acid residue.

[0160] L represents a leucine residue.

[0161] V represents a valine residue.

[0162] And W is a tryptophan residue.

[0163] This specification contains the contents described in the specification and / or drawings of Japanese Patent Application No. 2011-182539, which forms the basis of the priority claim of this application.

[0164] Invention Effects

[0165] The human IgG-binding peptide of the present invention has the following advantages: it exhibits higher selectivity for IgG compared to IgA, IgM, and IgE, and can bind to human IgG. This means that IgG can be selectively isolated from, for example, human serum. Attached Figure Description

[0166] Figure 1 This indicates the binding specificity of human IgG to phage clones using ELISA;

[0167] Figure 2 The sequence (A) represents Lib-A and the sequence (B) represents the peptide derived from that sequence;

[0168] Figure 3 Represents the sequence of Lib-B;

[0169] Figure 4 Represents a sequence of Lib-C;

[0170] Figure 5 This indicates the results of the binding analysis of human IgG (left) and human IgA (right) using surface plasmon GFc-C35-3 / 15 (T5A, T7H, N8R, N10E) peptide (A) and GFc-C35-3 / 15 (T5A, Y6W, T7H, N8R, N10E) peptide (B).

[0171] Figure 6 Represents the sequence of Lib-D;

[0172] Figure 7This indicates the frequency of amino acid occurrences at each site based on the peptide sequence obtained from Lib-D;

[0173] Figure 8 This indicates the results of IgG purification from human serum using a column immobilized with IgG-binding peptides;

[0174] Figure 9 This represents the SDS-PAGE results of elution fraction D purified from human serum using a column immobilized with IgG-binding peptides. Each lane represents one of the following samples: 1: Label, 2: IgG, 3: HAS, 4: Serum, 5: Elution fraction D. Detailed Implementation

[0175] In this invention, the peptides that specifically or selectively bind to human IgG were isolated from a newly designed and constructed library containing one disulfide bond within the molecule, constructed using the T7 phage display system (Sakamoto, K., Ito, Y., Hatanaka, T., Soni, PB, Mori, T., and Sugimura, K. (2009) The Journal of Biological Chemistry 284(15), 9986-9993). The two specific clones obtained showed homology to their shared sequences, and synthetic peptides prepared by various substitutions or deletions of these sequences exhibited specificity for IgG. Identification of the residues essential for IgG binding to these peptides made it possible to apply them to affinity enhancement methods and to purify IgG from human serum. The IgG-binding peptides of this invention are the smallest peptides, as small as 13 residues, thus enabling the construction of IgG purification systems based on low-cost peptides.

[0176] The present invention will now be described in further detail.

[0177] Specifically, the IgG binding peptide of the present invention, the purification and analytical methods of IgG using the peptide, and the kit for such IgG purification or detection will be described.

[0178] IgG binding peptide

[0179] The peptides of the present invention were screened from a phage library containing a large number of random peptides as peptides that specifically or selectively bind to human IgG.

[0180] The human IgG used in this instruction manual refers to IgG1, IgG2, IgG3, and IgG4.

[0181] That is, the peptide of the present invention, as a primary structure in a broad sense, is characterized by containing an amino acid sequence of 13 to 17 amino acid residues as shown in Formula I below, and is capable of binding to human IgG:

[0182] (X 1-3 )-C-(X2)-HRG-(Xaa1)-LVWC-(X 1-3 (I)

[0183] In the formula, X can be any amino acid residue other than cysteine.

[0184] C is a cysteine ​​residue.

[0185] H stands for histidine residue.

[0186] R represents an arginine residue.

[0187] G stands for a glycine residue.

[0188] Xaa1 is a glutamic acid residue or an asparagine residue.

[0189] L represents a leucine residue.

[0190] V is a valine residue, and

[0191] W represents a tryptophan residue.

[0192] In the above formula, X at the N-terminus or C-terminus 1-3 The symbol X1 refers to any independent amino acid residue X other than 1 to 3 consecutive cysteine ​​residues (C or Cys), whose constituent amino acid residues may be the same or different, but preferably consist of a sequence of 3 different residues. Similarly, X2 also refers to any independent amino acid residue X other than 2 consecutive cysteine ​​residues (C or Cys), whose constituent amino acid residues may be the same or different, but preferably consist of a sequence of 2 different consecutive amino acid residues.

[0193] The two cysteine ​​residues in Formula I can form a disulfide bond, thus forming a cyclic peptide. Typically, peptides of Formula I form disulfide bonds.

[0194] In the amino acid sequence of the peptide of Formula I, the peptides of Formula I' and Formula I'', which further specify amino acid residue X, are shown below.

[0195] That is, the peptide represented by Formula I' is characterized by containing the amino acid sequence of 13 to 17 amino acid residues as shown in Formula I', and is capable of binding to human IgG:

[0196] (X 1-3 )-C-(X1)-YHRGNLVWC-(X 1-3) (I')

[0197] In the formula, X can be any amino acid residue other than cysteine.

[0198] C is a cysteine ​​residue.

[0199] Y represents a tyrosine residue.

[0200] H stands for histidine residue.

[0201] R represents an arginine residue.

[0202] G stands for a glycine residue.

[0203] N represents an asparagine residue.

[0204] L represents a leucine residue.

[0205] V is a valine residue, and,

[0206] W represents a tryptophan residue.

[0207] The peptide represented by Formula I'' is characterized by containing the following amino acid sequence of Formula I'' consisting of 13 to 17 amino acid residues, and is capable of binding to human IgG:

[0208] (X 1-3 )-CA-(X1)-HRGELVWC-(X 1-3 ) (I'')

[0209] In the formula, X can be any amino acid residue other than cysteine.

[0210] C is a cysteine ​​residue.

[0211] A is an alanine residue.

[0212] H stands for histidine residue.

[0213] R represents an arginine residue.

[0214] G stands for a glycine residue.

[0215] E represents a glutamic acid residue.

[0216] L represents a leucine residue.

[0217] V is a valine residue, and

[0218] W represents a tryptophan residue.

[0219] In addition, in the amino acid sequence of the peptide of Formula I, the peptide of Formula II, which specifies amino acid residue X, is shown below.

[0220] That is, the peptide represented by Formula II is characterized by containing the amino acid sequence of 13 to 17 amino acid residues shown in Formula II, and is capable of binding to human IgG:

[0221] (X 1-3 )-C-(Xaa2)-(Xaa3)-HRG-(Xaa1)-LVWC-(X 1-3 (II)

[0222] In the formula, X can be any amino acid residue other than cysteine.

[0223] C is a cysteine ​​residue.

[0224] H stands for histidine residue.

[0225] R represents an arginine residue.

[0226] G stands for a glycine residue.

[0227] Xaa1 is a glutamic acid residue or an asparagine residue.

[0228] L represents a leucine residue.

[0229] V represents a valine residue.

[0230] W represents a tryptophan residue.

[0231] Xaa2 is an alanine residue, a serine residue, or a threonine residue, and

[0232] Xaa3 is a tyrosine residue or a tryptophan residue.

[0233] In the amino acid sequences of the peptides of Formula I', Formula I'' and Formula II mentioned above, when there are 17 amino acid residues, the first and second amino acid residues X from the N-terminus and the 16th and 17th amino acid residues can be omitted, and such peptides are composed of 13 amino acid lengths.

[0234] The phrase "when set to 17 amino acid residues" as used in this specification is a convenient term used when naming the amino acid residues of a peptide by amino acid numbering, in order to number the 1st to the 17th residues sequentially from the N-terminus of the 17th residues, which is the longest amino acid.

[0235] Furthermore, in the amino acid sequence of the peptide of Formula I, the peptide of Formula III, which specifies amino acid residue X, is shown below.

[0236] The peptide represented by Formula III is characterized by containing the following amino acid sequence of Formula III consisting of 13 to 17 amino acid residues, and is capable of binding to human IgG:

[0237] (X1-3 )-CAYHRGELVWC-(X 1-3 (III)

[0238] In the formula, X can be any amino acid residue other than cysteine.

[0239] C is a cysteine ​​residue.

[0240] A is an alanine residue.

[0241] Y represents a tyrosine residue.

[0242] H stands for histidine residue.

[0243] R represents an arginine residue.

[0244] G stands for a glycine residue.

[0245] E represents a glutamic acid residue.

[0246] L represents a leucine residue.

[0247] V is a valine residue, and

[0248] W represents a tryptophan residue.

[0249] In the amino acid sequence of the peptide of Formula III above, when it is set to 17 amino acid residues, the first, second, 16th and 17th amino acid residues X from the N-terminus can be omitted, and such a peptide is composed of 13 amino acid lengths.

[0250] Furthermore, when the amino acid sequence of the aforementioned peptides contains 17 amino acid residues other than cysteine ​​(C), the amino acid residues from the N-terminus, numbered 1 to 3, 5, 6, and 15 to 17, are preferably selected from the following residues. Wherein, each capital letter represents a single-letter symbol for an amino acid:

[0251] The first amino acid residue is either S, G, F, or absent;

[0252] The second amino acid residue is either D, G, A, S, P, or absent;

[0253] The third amino acid residue = S, D, T, N, E, or R;

[0254] The 5th amino acid residue = A or T;

[0255] The 6th amino acid residue = Y or W;

[0256] The 15th amino acid residue = S, T, or D;

[0257] The 16th amino acid residue = H, G, Y, T, N, D, F or none;

[0258] The 17th amino acid residue can be Y, F, H, M, or absent.

[0259] The 5th amino acid residue = A or T;

[0260] The 6th amino acid residue = Y or W;

[0261] In the amino acid sequence of the peptide of Formula I, the peptide of Formula IV, which further specifies amino acid residue X, is shown below.

[0262] The peptide represented by Formula IV is characterized by containing an amino acid sequence of 13 amino acid residues as shown in Formula IV, and is capable of binding to human IgG.

[0263] DC-(Xaa2)-(Xaa3)-HRG-(Xaa1)-LVWCT (IV)

[0264] (D represents an aspartic acid residue)

[0265] C is a cysteine ​​residue.

[0266] H stands for histidine residue.

[0267] R represents an arginine residue.

[0268] G stands for a glycine residue.

[0269] Xaa1 is a glutamic acid residue or an asparagine residue.

[0270] L represents a leucine residue.

[0271] V represents a valine residue.

[0272] W represents a tryptophan residue.

[0273] T stands for a threonine residue.

[0274] Xaa2 is an alanine residue or a threonine residue, and

[0275] Xaa3 is a tyrosine or tryptophan residue.

[0276] Examples of several peptides of Formula I are given in 1) to 17) below, but the examples are not limited thereto. Such peptides exhibit exceptionally high binding specificity or selectivity for human IgA compared to other types of immunoglobulins:

[0277] 1)DCTYHRGNLVWCT (SEQ ID NO: 47)

[0278] 2)DCAYHRGNLVWCT (SEQ ID NO: 48)

[0279] 3)DCTYHRGELVWCT (SEQ ID NO: 50)

[0280] 4)DCAYHRGELVWCT (SEQ ID NO: 52)

[0281] 5) DCAWHRGELVWCT (SEQ ID NO: 53)

[0282] 6)DCAYHRGELVWCT (SEQ ID NO: 55)

[0283] 7)GPDCAYHRGELVWCTFH (SEQ ID NO: 56)

[0284] 8)RCAYHRGELVWCS (SEQ ID NO: 57)

[0285] 9)GPRCAYHRGELVWCSFH (SEQ ID NO: 58)

[0286] 10)SPDCAYHRGELVWCTFH (SEQ ID NO: 100)

[0287] 11)GDDCAYHRGELVWCTFH (SEQ ID NO: 101)

[0288] 12) GPSCAYHRGELVWCTFH (SEQ ID NO: 102)

[0289] 13)GPDCAYHRGELVWCSFH (SEQ ID NO: 103)

[0290] 14)GPDCAYHRGELVWCTHH (SEQ ID NO: 104)

[0291] 15)GPDCAYHRGELVWCTFY (SEQ ID NO: 105)

[0292] 16)SPDCAYHRGELVWCTFY (SEQ ID NO: 106)

[0293] 17)SDDCAYHRGELVWCTFY (SEQ ID NO: 107).

[0294] Furthermore, the peptide of the present invention, as a primary structure in a broad sense, is characterized by containing an amino acid sequence of 13 amino acid residues as shown in Formula V below, and is capable of binding to human IgG:

[0295] DC-(Xaa1)-(Xaa2)-(Xaa3)-(Xaa4)-G-(Xaa5)-L-(Xaa6)-WCT (V)

[0296] In the formula,

[0297] D stands for aspartic acid residue.

[0298] C is a cysteine ​​residue.

[0299] G stands for a glycine residue.

[0300] L represents a leucine residue.

[0301] W represents a tryptophan residue.

[0302] T stands for a threonine residue.

[0303] Xaa1 is an alanine residue, a serine residue, or a threonine residue.

[0304] Xaa2 is a tryptophan residue or a tyrosine residue.

[0305] Xaa3 is a histidine residue, arginine residue, serine residue, or threonine residue.

[0306] Xaa4 is an asparagine residue or an arginine residue.

[0307] Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue.

[0308] Furthermore, Xaa6 is an isoleucine residue or a valine residue.

[0309] The two cysteine ​​residues of formula V can form a disulfide bond, thus forming a cyclic peptide. Typically, peptides of formula V form disulfide bonds.

[0310] The following examples (10) to 21) illustrate several specific examples of peptides of formula V, but are not limited to these examples. Such peptides exhibit exceptionally high binding specificity or selectivity for IgA compared to other types of immunoglobulins:

[0311] 10)DCTYTNGNLVWCT (SEQ ID NO: 29)

[0312] 11)DCAYTNGNLVWCT (SEQ ID NO: 31)

[0313] 12)DCSYTNGNLVWCT (SEQ ID NO: 32)

[0314] 13)DCTWTNGNLVWCT (SEQ ID NO: 34)

[0315] 14)DCTYHNGNLVWCT (SEQ ID NO: 35)

[0316] 15)DCTYRNGNLVWCT (SEQ ID NO: 36)

[0317] 16)DCTYSNGNLVWCT (SEQ ID NO: 37)

[0318] 17)DCTYTRGNLVWCT (SEQ ID NO: 39)

[0319] 18)DCTYTNGELVWCT (SEQ ID NO: 40)

[0320] 19)DCTYTNGRLVWCT (SEQ ID NO: 41)

[0321] 20)DCTYTNGDLVWCT (SEQ ID NO: 42)

[0322] 21) DCTYTNGNLIWCT (SEQ ID NO: 45).

[0323] As described above, the peptide of the above-described formula involved in this invention is characterized in that each amino acid sequence has two separated cysteine ​​(C) residues, and the cysteine ​​residues are arranged in such a way that disulfide bonds can be formed between these cysteine ​​residues. Preferably, the peptide is a cyclic peptide formed by two cysteine ​​residues forming disulfide bonds. One or two arbitrary amino acid residues other than cysteine ​​may be present on the N-terminal and C-terminal sides of each cysteine ​​residue. In the case where one or two amino acid residues are present on the N-terminal and C-terminal sides of each cysteine ​​residue, it is set to 17 amino acid residues, with the first to second and 16th to 17th amino acid residues from the N-terminus being the residues exemplified above.

[0324] The binding affinity of the peptide of the present invention to human IgG is about 10 times higher than that of other human immunoglobulins (IgA, IgE, IgM), preferably about 50 times higher, and more preferably about 200 times higher. The dissociation constant (Kd) of the peptide of the present invention for binding to human IgG can be determined by surface plasmon resonance spectroscopy analysis (e.g., using a BIACORE system), and is, for example, 1 × 10⁻⁶. -1 M ~ below 1×10 -3 M, preferably less than 1×10 -4 M, more preferably less than 1×10 -5 M.

[0325] The peptides of this invention can be manufactured using conventional liquid-phase synthesis, solid-phase synthesis, or peptide synthesis using automated peptide synthesizers (Kelley et al., Genetics Engineering Principles and Methods, Setlow, J.Keds., Plenum Press NY. (1990) Vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide S y nthesis (1989) WH Freeman Co.; Houghten, Proc. Natl. Acad. Sci. USA (1985) 82: p. 5132; "New Biochemistry Experiment Lectures 1 Protein IV" (1992) edited by the Japanese Society for Biochemistry, Tokyo Chemical Association). Alternatively, the peptide can be manufactured by gene recombination of the nucleic acid encoding the peptide of the present invention or by phage display. For example, DNA encoding the amino acid sequence of the peptide of the present invention can be introduced into an expression vector and cultured in a host cell to produce the target peptide. The manufactured peptide can be recovered or purified by conventional methods such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reversed-phase column chromatography, HPLC, ammonium sulfate fractionation, ultrafiltration, immunosorbent assay, etc.

[0326] Peptide synthesis involves preparing amino acids with protected functional groups other than the α-amino and α-carboxyl groups, through a peptide bond formation reaction between the α-amino and α-carboxyl groups of each amino acid. Typically, the carboxyl group of the amino acid residue at the C-terminus of the peptide is attached to a solid phase via a suitable spacer or linker. The protecting group at the amino terminus of the resulting dipeptide is selectively removed, forming a peptide bond with the α-carboxyl group of the next amino acid. This process is repeated continuously to produce peptides with protected side groups. Finally, all protecting groups are removed, and the peptide is separated from the solid phase. Details regarding the types of protecting groups, protection methods, and peptide bond formation are described in the aforementioned literature.

[0327] The gene recombination method includes inserting DNA encoding the peptide of the present invention into a suitable expression vector, introducing the vector into a suitable host cell, culturing the cell, and recovering the target peptide from the intracellular or extracellular fluid. The vector is not limited and can be, for example, a plasmid, bacteriophage, granule, phage particle, virus, etc. Plasmid vectors are not limited and can include: plasmids derived from *Escherichia coli* (e.g., pET22b(+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from *Bacillus subtilis* (e.g., pUB110, pTP5, etc.), plasmids derived from yeast (e.g., YEp13, YCp50, etc.), etc. Phage vectors are not limited, but examples include: T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc. (Novagen)), and λ phage vectors (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). Viral vectors are not limited, but examples include: retroviruses, adenoviruses, adeno-associated viruses, vaccinia virus, Sendai virus, and other animal viruses; baculoviruses and other insect viruses. Glumoid vectors are not limited, but examples include: Lorist6, Charomid9-20, Charomid9-42, etc. Phage particle vectors are not limited, but known examples include pSKAN, pBluescript, pBK, pComb3H, etc. The vector may contain regulatory sequences that express target DNA, selection markers for panning vectors containing target DNA, and multiple cloning sites for inserting target DNA. Such regulatory sequences may include promoters, enhancers, terminators, SD sequences or ribosome binding sites, replication initiation regions, and multiple A sites. Additionally, selection markers may include, for example, ampicillin resistance genes, neomycin resistance genes, kanamycin resistance genes, and dihydrofolate reductase genes. Host cells used for vector introduction include *E. coli* and *Bacillus subtilis* cells, yeast cells, insect cells, animal cells (e.g., mammalian cells), and plant cells. Transformation or transfection of these cells may be performed using methods such as calcium phosphate method, electroporation, liposome transfection, particle gun method, and PEG method. The method for culturing the transformed cells can be based on methods commonly used in the culture of the host organism. For example, in the culture of microorganisms such as *E. coli* and yeast cells, there are carbon sources, nitrogen sources, and inorganic salts that the host microorganisms can assimilate. To facilitate the recovery of the peptides of the present invention, it is preferable to secrete the peptides generated through expression extracellularly. Therefore, DNA encoding a peptide sequence that allows the peptide to be secreted from the cell is bound to the 5' end of the DNA encoding the target peptide. The fusion peptide transferred to the cell membrane is cleaved by a signal peptidase, thereby releasing the target peptide into the culture medium.Alternatively, the target peptide accumulated within the cell can be recovered. In this case, the cell is physically or chemically destroyed, and protein purification techniques are used to recover the target peptide.

[0328] Therefore, the present invention further relates to a nucleic acid encoding the peptide of the present invention. Here, nucleic acid includes DNA or RNA (e.g., mRNA).

[0329] To enable the detection of IgG, the peptides of this invention can also be labeled. Labeling is not limited, and may include, for example, fluorescent dyes, chemiluminescent dyes, enzymes, radioisotopes, fluorescent proteins, biotin, etc. Preferred examples of labeling include fluorescein, FITC and other fluorescein derivatives, rhodamine derivatives such as rhodamine and tetramethylrhodamine, and fluorescent dyes such as Texas red.

[0330] The peptide of this invention can be fused with any protein. The protein can be used as a label, as long as it is a fluorescent protein such as GFP (green fluorescent protein), an enzyme such as peroxidase, etc. In this case, the peptide of this invention and the protein can be made into a fusion protein via gene recombination method using an appropriate linker, as needed. At this time, the peptide of this invention should be prepared into a fusion protein in a manner that does not impair its binding affinity to human IgG.

[0331] The peptides of the present invention can be further immobilized on a solid phase that can be packed into an affinity column in a manner that enables the separation, purification, and analysis of human IgG.

[0332] There are no limitations on the solid phase suitable for peptide immobilization, and examples include: polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, styrene-butadiene copolymer, (meth)acrylate polymers, fluoropolymers, silica gel, cross-linked dextran, polysaccharides, agarose and other polysaccharides, glass, metals, magnetic materials, and combinations thereof. The shape of such a solid phase can be any shape, such as a disc, sphere, fiber, particle, rod, plate, container, box, microplate, test tube, membrane (membrane or thin film), gel, sheet, etc. Specifically, examples include: magnetic beads, glass beads, polystyrene beads, agarose beads, silica gel beads, polysaccharide beads, polystyrene plates, glass plates, polystyrene tubes, etc. The peptide immobilization of the present invention on these solid phases can be performed using methods known to those skilled in the art, such as physical adsorption, covalent bonding, ionic bonding, etc. Immobilization is preferably achieved through covalent bonding, giving the solid phase surface chemical functional groups (e.g., hydroxyl, amino, N-hydroxysuccinimide, etc.), preferably chemical functional groups containing alkylene chains with about 4 to 20 carbon atoms as spacers, which then react chemically with the carboxyl terminus of the peptide to form ester or amide bonds, etc. The solid phase immobilized with the peptide of the present invention is packed into columns such as affinity chromatography columns or HPLC columns, and can be used for the detection, purification, or separation of human IgG.

[0333] Purification method of IgG

[0334] The present invention further provides a method for purifying IgG, comprising binding the peptide or immobilized peptide of the present invention to IgG and releasing and recovering the bound IgG.

[0335] The solid phase immobilized with the peptides of the present invention is packed into an affinity chromatography column, HPLC column, or similar column, equilibrated with a suitable buffer, and then subjected to a liquid containing human IgG at a low temperature of room temperature to 0°C, preferably about 10°C to 0°C, and more preferably about 4°C, to bind the human IgG to the peptides on the solid phase. For example, in the case of separating IgG from serum, a buffer with a neutral pH range, such as pH 6.0 to 7.5, can be used for column loading and binding. Elution can be performed by flowing a buffer with an acidic pH range, such as pH 2 to 4 (e.g., 0.2M glycine-HCl buffer containing 0.3M NaCl at pH 3.5 to 2.5) through the column.

[0336] Whether IgG has been recovered can be determined, for example, by electrophoresis followed by Western blotting using anti-human IgG antibodies. The electrophoresis conditions are as follows: SDS-PAGE using a 5–20% acrylamide gradient gel can be performed. The Western blotting conditions are as follows: the electrophoretically deposited protein can be transferred to a PVDF membrane, blocked with skim milk, and then detected using anti-human IgG α-chain goat antibody and HRP-labeled anti-goat IgG mouse antibody.

[0337] The method of the present invention can be used to obtain IgG-enriched fractions in the purification of IgG from IgG-containing products generated by various methods. Therefore, the method of the present invention is preferably used in column chromatography methods such as affinity chromatography and HPLC. In addition to such chromatographic methods, conventional purification techniques for combined proteins can also be used when purifying IgG, such as gel filtration chromatography, ion exchange column chromatography, reversed-phase column chromatography, ammonium sulfate fractionation, ultrafiltration, etc.

[0338] IgG analysis method

[0339] The present invention further provides a method for detecting IgG, comprising binding IgG in a sample to the peptide or immobilized peptide of the present invention described above to detect the bound IgG. Here, the detection includes either qualitative or quantitative analysis.

[0340] The detection of IgG can be performed as follows: using a suitable buffer solution, the sample is bound to a membrane or polystyrene plate, etc., so that it comes into contact with the labeled peptide of the present invention, and after washing as needed, the labeling level is qualitatively or quantitatively determined.

[0341] Alternatively, when using an HPLC column immobilized with the peptide of the present invention as described above, a sample containing human IgG is injected into the column, a binding buffer is passed through to bind the human IgG to the peptide, and the protein detected under fluorescence at 350 nm caused by excitation light at an absorbance of 280 nm, for example, is recorded. The IgG is eluted from the column with an elution buffer (for example, gradient elution in 0.1 M glycine hydrochloride buffer containing 0.15 M NaCl at pH 2.5). The qualitative and quantitative analysis of IgG can be performed by observing the peaks and peak areas that appear.

[0342] Reagent kit and column

[0343] The present invention further provides a kit for the analysis (qualitative, quantitative, etc.) or purification of human IgG, which contains at least one of the peptides or immobilized peptides of the present invention described above.

[0344] The various peptides or immobilized peptides contained in the kit of the present invention are housed in separate containers. Additionally, if necessary, the kit may also include instructions for use that describe the analytical and purification steps for human IgG. Furthermore, the kit may also contain reagents and buffers required for analysis, immobilized peptide packed columns, etc.

[0345] The present invention further provides an IgG separation column containing the immobilized peptides of the present invention described above.

[0346] The aforementioned IgG separation columns are columns used for separating IgG. Specifically, they include chromatographic columns, high-performance liquid chromatography (HPLC) columns, and other columns used for the analysis or purification / separation of IgG. There are no particular limitations on the column size; it can be varied depending on the intended use (analysis, purification / separation), application (loading), or injection volume. Furthermore, the column material can be metal, plastic, glass, or other commonly used column materials.

[0347] The column described above can be manufactured by tightly filling the column with the immobilized peptide (dry or wet state) of the present invention prepared according to the above method.

[0348] Example

[0349] The present invention will be further described in detail below with reference to specific embodiments, but the scope of the present invention is not limited to these embodiments.

[0350] To isolate human IgG-specific phages from a random peptide library with a circular structure formed by two Cys, constructed using the T7 phage display method, the following biopanning method was used.

[0351] That is, 5 × 10⁻⁶ PBS containing 0.5% BSA and 0.1 μM of type II IgG binding peptide K6R (J. Biol. Chem. 284, 9986, 2009) 10 PFU T7 phage library (X3CX8CX3, X3CX9CX3X3CX) 10 An equal volume mixture of CX3 was added to the wells of a 96-well microplate (Nunc, Maxisorp) coated with human IgG-Fc (from human plasma, Athens Research & Technology, Athens, GA, USA) (1 μg / 100 μl / well) and blocked with 0.5% BSA, and allowed to react for 1 hour. After removing the supernatant, the phage solution was washed 10 times with PBS containing 0.1% Tween. E. coli BLT5615 (Novagen) culture medium (300 μl) was added to infect the phage, and the plate was incubated with 3 ml of E. coli culture medium at 37°C until proliferation and lysis. The phage was recovered from the lysed culture medium using phage precipitation with polyethylene glycol according to standard methods. The obtained phage was dissolved in PBS, filtered through a 0.45 μm filter, and used for the following panning. By performing four rounds of panning, including the above steps, IgG-specific phage was concentrated.

[0352] The binding specificity of bacteriophages obtained after four rounds of screening to various IgGs was investigated using ELISA. The results are as follows: Figure 1 As shown, it not only binds to human IgG, but also exhibits binding activity with IgG from rabbits, goats, and mice.

[0353] Therefore, the peptide motifs displayed by the obtained phages were analyzed to determine the amino acid sequences (Table 1).

[0354] [Table 1]

[0355] Comparison of peptide sequences displayed by IgG-binding phages obtained from random peptide libraries

[0356] 1******8-9*******17 GFc-A2 SFTCAYDRDGNLVWCTHS SEQ ID NO: 1 1 / 30 Fx-B17 SSDCTYQR-GELVWCTHL SEQ ID NO: 2 1 / 30 GFc-C3 PGECTKHM-GELVWCVSK SEQ ID NO: 3 1 / 80 GFc-C35 GPDGTYTN-GNLVWCTFH SEQ ID NO: 4 2 / 80 GFc-C65 KPRCSYLR-CQLVWCLHS SEQ ID NO: 5 2 / 80 ***C*****G*LVWC***

[0357] The amino acid numbering of the peptides is based on the length of the X3CX9CX3 peptide library, numbered from 1 to 17 starting from the N-terminus.

[0358] Peptide synthesis was performed on GFc-C35, which has strong binding activity, and affinity was evaluated using surface plasmon resonance (SPR) analysis. The results showed that the Kd value was 14 μM, indicating low affinity. For use as an affinity ligand, it is necessary to enhance the affinity.

[0359] Therefore, firstly, focusing on the region sandwiched between two Cys molecules, the completely conserved Gly9, Leu11, Val12, Trp13, and Thr15 molecules in GFc-C35 were immobilized. Then, a library (library A: Lib-A) was constructed by randomizing other sites using NNK-mixed nucleotides. Figure 2 (A)) The sequence of peptides obtained through biopanning was used to evaluate the preferential binding characteristics of amino acids (wherein, the amino acid sequences of peptides obtained from the library showed almost no common characteristics at position 1, and were therefore removed from the library). After biopanning human antibodies using library A, the sequences of the resulting phage peptides and the amino acids found at each amino acid site are shown below. Figure 2 (B) At position 5, only Thr, Ser, and Ala with small side chains were found. At position 6, Trp was the most abundant, and Tyr and Phe, which have aromatic ring side chains, were also present. At position 7, His was the most abundant, with some Ser and Trp also found. Furthermore, Arg, Leu, and Met were abundant at position 8. Finally, position 10 was occupied by hydrophilic amino acids, with Arg being the most abundant. Figure 2 ).

[0360] The above results clarified the characteristics of the side chains of important sites in IgG binding when Gly9, Leu11, Val12, Trp13 and Thr15 are immobilized. However, in order to confirm the importance of conserved residues and screen for peptides with stronger binding forces, library B:Lib-B was designed again.

[0361] That is, such as Figure 3 As shown, while maintaining the characteristics of the cloned peptide sequences obtained in Lib-A, mutations were introduced into the completely conserved amino acids (Leu11, Val12, Trp13, and Thr15) in Table 1 by introducing amino acids with similar side chains to construct a library. Gly9 was considered important for maintaining the peptide's stereostructure and was therefore fixed. Additionally, the two outermost amino acid residues of Cys were removed from the library construction, and only the original GFc-C35 peptide sequences (Asp3 and Thr15) were added. Using the constructed library, biopanning was performed under strict washing conditions, and ELISA screening analysis revealed cloned peptide sequences with strong binding activity.

[0362] The resulting sequences are shown in Table 2.

[0363] [Table 2]

[0364] 39 1-T3331 DC SYRF CELVW CT SEQ ID NO: 6 2-T3338 DC SYHF GELVW CT SEQ ID NO: 7 3-T33313 DC AFHL GHLVW CT SEQ ID NO: 8 4-T33314 DC AFHR CDLVW CT SEQ ID NO: 9 5-T33315 DC AFHF GDLVW CT SEQ ID NO: 10 6-T33320 DC TYHF GKLVW CT SEQ ID NO: 11 1-T33324 DC AFHL CELVR CT SEQ ID NO: 12 8-T333317 DC TWKF GDLIW CT SEQ ID NO: 13 9-T33339 DC AYHL GQLVR CT SEQ ID NO: 14 10-T33341 DC SFHL GDLVW CT SEQ ID NO: 15 11-T32213 DCSYHL GDYVW CT SEQ ID NO: 16 12-T32222 DC SWHM GQLIW CT SEQ ID NO: 17 AYHF DLVW SF L E TW

[0365] Results: At position 5, Ala was the most abundant, but no significant preference was found compared to Ser and Thr. At position 6, Tyr and Phe were preferred, replacing the prominent Trp in Lib-A. At position 7, His was overwhelmingly abundant despite the introduction of various amino acids into the library. At position 8, Phe and Leu were abundant, replacing Arg and Leu in Lib-A. At position 10, the acidic amino acids Asp and Glu were abundant, but a few positively charged Lys, His, and Gln were also found. On the other hand, as expected from the sequences of the initially isolated clones, Leu11, Val12, and Trp13 were largely conserved, but very rarely were Tyr replaced at position 11, Ile at position 12, and Arg at position 13.

[0366] The high frequency of these amino acids is believed to reflect the contribution of the side chains of each amino acid to IgG binding. Table 3 summarizes the amino acids found at high frequency at each site in the cloned peptide sequences obtained from the original libraries (Library O: Lib-O) and libraries A and B (Lib-A,B).

[0367] [Table 3]

[0368] The amino acids that appear at high frequency in the peptide sequences of phages obtained by panning various libraries.

[0369] 5 6 7 8 9 10 11 12 13 15 16 Lib0 Y R G L V W T H Lib A SAT WFY HSW RLM - REN - - - - Lib B AST YEW H FL - DE L V W - Lib C SA WFY H L(R) - Q ML V(I) W TS

[0370] In the table, - indicates immobilized amino acids, namely Gly9, Leu11, Va112, Trp13, and Thr15.

[0371] Based on this information, sequences were further added to the outer sides of the Cys on both sides to construct a randomized library (Lib-C). Figure 4 In this library, the sequence inside Cys contains amino acids that appear frequently in Lib-A and B, while the residues on both sides of Cys are randomized in a manner that contains a completely random sequence of mixed nucleotides using NNK or similar amino acids found in Lib-O or Lib-A. Regarding the library structure, Thr was originally intended to be placed at position 6, but if Thr were added to the nucleotide mixture, the number of amino acids would become too large. Therefore, a hydrophobic and / or aromatic amino acid was added instead of Thr. In addition, Ser, Arg, Trp, etc., were originally intended to be placed at position 7, but His is overwhelmingly abundant, so His was added. Tyr and Gln were added as controls.

[0372] Using this library, clones with high binding affinity were selected again through panning, and the results of sequence analysis are shown in Table 4.

[0373] [Table 4]

[0374] GFc-C35 GPDC TYTNC NlVWC TFH SEQ ID NO: 4 T6-1 RGC SYHLG QLVWC TAV SEQ ID NO: 18 T6-2 VKC SWHLG QMVWC TSN SEQ ID NO: 19 T6-7 ANC SWHLG DMVWC STI SEQ ID NO: 20 T6-15 VKC SWHLG QMVWC SNS SEQ ID NO: 21 T6-16 VKC SWHLG QMVWC SNS SEQ ID NO: 22 T6-20 LNC AFHRC RLVWC TDL T6-26 SEQ ID NO: 23 [[ID=9 T6-33 ​ ​ T6-41 ​ ​ T6-43 ​ ​ ​ ​ F

[0375] Results: No characteristic amino acids were found at positions 2 and 3 outside the N-terminal Cys, but Thr or Ser appeared multiple times at position 15 outside the C-terminal Cys. However, no characteristic amino acids were found at positions 16 and 17. Regarding the internal sequence, a sequence was obtained that did not contradict the results obtained from Lib-O, A, and B (Table 3).

[0376] Next, the effects of synthetic peptides on the introduction of amino acids at high frequencies at sites believed to enhance binding were evaluated and verified.

[0377] To narrow down the region of amino acids used for validation, peptides with one (GFc-C35-2 / 16) or two (GFc-C35-3 / 15) amino acids deleted from both ends of the GFc-C35 sequence were synthesized. The effect of the synthesized GFc-C35-3 / 15 peptide on affinity induced by amino acid mutations was evaluated using this synthetic peptide as a benchmark. Specifically, peptides with amino acids replaced by those observed at high frequency or in a subset from phage libraries were synthesized, and their binding was analyzed to evaluate the contribution of each amino acid replacement to affinity. The results are shown in Table 5.

[0378] [Table 5]

[0379] Affinity evaluation of amino acid substitution in synthetic skin

[0380] ​ Sequence Kd (μM) GFc-C35 GPDCTYTNGNLVWCTFH SEQ ID NO: 4 14 GFc-C35-2 / 16 PDCTYTNGNLVWCT SEQ ID NO: 28 25 GFc-C35-3 / 15 DCTYTNGNLVWCT SEQ ID NO: 29 130 GFc-C35-3 / 15 (D3R) RCTYTNGNLVWCT SEQ ID NO: 30 69 GFc-C35-3 / 15 (T5A) DCAYTNGNLVWCT SEQ ID NO: 31 61 GFc-C35-3 / 15 (T5S) DCSYTNGNLVWCT SEQ ID NO: 32 120 GFc-C35-3 / 15 (Y6F) DCTFTNGNLVWCT SEQ ID NO: 33 2100 GFc-C35-3 / 15 (Y6W) DCTWTNGNLVWCT SEQ ID NO: 34 50 GFc-C35-3 / 15 (T7H) DCTYHNGNLVWCT SEQ ID NO: 35 3 GFc-C35-3 / 15 (T7R) DCTYRNGNLVWCT SEQ ID NO: 36 83 GFc-C35-3 / 15 (T7S) DCTYSNGNLVWCT SEQ ID NO: 37 26 GFc-C35-3 / 15 (N8L) DCTYTLGNLVWCT SEQ ID NO: 38 260 [[ID= ​ ​ 8 ​ ​ ​ 26 ​ ​ ​ 42 ​ ​ ​ 80 ​ ​ ​ 160 ​ ​ ​ 280 ​ ​ ​ 12 ​ ​ ​ 34

[0381] Affinity analysis was performed on peptides with one or two residues deleted from each end (GFc-C35-2 / 16) (GFc-C35-3 / 15). The results showed that the Kd values ​​for each residue were 25 μM and 130 μM, respectively, compared to 14 μM for GFc-C35, indicating increased affinity and decreased affinity. These results clearly demonstrate that the residues at both ends (positions 1, 2, 16, and 17) facilitate binding.

[0382] Regarding Asp3, Arg was found to be common in Lib-0 and Lib-C (Tables 1 and 4). Comparison of the affinity of the substituted peptide (GFc-C35-3 / 15(D3R)) and the original peptide (GFc-C35-3 / 15) revealed an increase in affinity.

[0383] Regarding Thr5, almost only Ala, Thr, and Ser were found in Lib-A. Furthermore, no preference was found in any of the three amino acids in subsequent Lib-B and C (Table 3). Comparing the affinity of the two substituted peptides (GFc-C35-3 / 15(T5A) and GFc-C35-3 / 15(T5S)) with the original peptide (GFc-C35-3 / 15), the peptide substituted with Ala showed the highest affinity.

[0384] Regarding Tyr6, Trp, Tyr, and Phe were found in roughly equal proportions in Lib-A and B. However, after creating and evaluating their respective replacements, it was found that the affinity of GFc-C35-3 / 15(Y6F) was significantly reduced, while the affinity increased when replaced with Trp (GFc-C35-3 / 15(Y6W)).

[0385] Regarding Thr7, substitutions of His and Ser obtained from Lib-A were created and evaluated. In both cases, affinity was significantly improved, but in particular, substitution with His resulted in a significant increase in affinity (approximately 50-fold).

[0386] Regarding Asn8, substitutions were performed using Leu and Arg, which are abundant in Lib-O, Lib-A, and Lib-B. When Arg was substituted (GFc-C35-3 / 15(N8R)), a decrease in Kd value of approximately 18-fold was observed, with a significant increase in affinity. Conversely, substitution with Leu (GFc-C35-3 / 15(N8L)) significantly reduced affinity.

[0387] Regarding Asn10, a large number of Arg and Glu residues with opposite charges were found in Lib-A. In peptides incorporating this amino acid (GFc-C35-3 / 15(N10R) and GFc-C35-3 / 15(N10E)), the affinity was increased compared to the original peptide (GFc-C35-3 / 15). This result indicates that charged residues can increase binding activity. This result is also supported by the fact that when Asn10 is replaced with the uncharged Gln (GFc-C35-3 / 15(N10Q)), the affinity is approximately the same as the original peptide without any enhancement. On the other hand, some increase in affinity was also confirmed when Asn10 was replaced with Asp (GFc-C35-3 / 15(N10D)), but no increase in affinity was observed in the peptide replaced with Glu, which has a longer side chain (GFc-C35-3 / 15(N10E)). Therefore, it is shown that the introduced amino acid residues have side chains of a certain length in order to improve affinity.

[0388] Regarding Leu11, the Leu residue at position 11 is overwhelmingly abundant in Lib-O and Lib-B, and is considered an important residue for binding. However, Met is also found in Lib-C. Therefore, when Leu11 (GFc-C35-3 / 15(L11M)) is replaced with Met, a decrease in affinity (approximately 2-fold in terms of Kd value) is observed.

[0389] Regarding Val12, when it is replaced with Ile found in Lib-C, the Kd value is about 1 / 10 compared to the original peptide, and the affinity is improved.

[0390] Regarding Thr15, when it was replaced with Ser, which appears frequently in Lib-C, an increase in affinity was observed (approximately 1 / 4 in terms of Kd value) compared to the original peptide.

[0391] Based on the above research, the synthetic combinatorial predictions suggest peptides derived from amino acid substitutions that contribute to improved affinity (Table 6).

[0392] [Table 6]

[0393] The combination of amino acid substitutions leads to an increase in the affinity of G-binding skin.

[0394] ​ ​ ​ ​ ​ ​ 1.1 ​ ​ ​ 0.25 ​ ​ ​ 2.0 CFc-C35-3 / 15(T7H, @N8R,N10E) DCTYHRGELVWCT SEQ ID NO: 50 0.27 GFc-C35-3 / 15(T7H, U8R, V12I) DCTYHRGNLIWCT SEQ ID NO:51 4.8 GFc-C35-3 / 15(T5A, T7H, N8R, N10E) DCAYHRGELVWCT SEQ ID NO: 52 0.054 CFc-C35-3 / 15(T5A, Y6W, T7H, N8R, N10E) DCAWHRGELVWCT SEQ ID NO: 53 0.26 GFc-C35-3 / 15(D3R, T5A, T7H, N8R, N10E, T15S) RCAYHRGELVWCS SEQ ID NO: 54 0.0147

[0395] First, when the substitutions Thr7His and Asn8Arg (GFc-C35-3 / 15(T7H,N8R)) with the greatest effect were introduced, the Kd value decreased to 1.1 μM, indicating an enhanced affinity.

[0396] Furthermore, when one of each of the effective T5A, Y6W, N10E, and V12I from Table 5 was introduced into the peptide (GFc-C35-3 / 15(T7H, N8R, T5A) or GFc-C35-3 / 15(T7H, N8R, N10E)), no affinity enhancement was observed in peptides (GFc-C35-3 / 15(T7H, N8R, T5A) or GFc-C35-3 / 15(T7H, N8R, N10E)) introduced with T5A or N10E. However, affinity decreased in peptides (GFc-C35-3 / 15(T7H, N8R, Y6W) or GFc-C35-3 / 15(T7H, N8R, V12I)) introduced with Y6W or V12I. This indicates that even with the accumulation of various advantageous changes, additive properties may not hold due to the compensatory and interfering effects of their mutual interactions.

[0397] Next, surface plasmon resonance (SPR) analysis was performed on the peptides GFc-C35-3 / 15 (T5A, T7H, N8R, N10E) and GFc-C35-3 / 15 (T5A, Y6W, T7H, N8R, N10E) with high affinity enhancement.

[0398] The results are shown in Figure 5 .

[0399] Analysis of the sensor data showed that the affinity (Kd value) of GFc-C35-3 / 15 (T5A, T7H, N8R, N10E) was 54 nM, approximately 1 / 24th of the Kd value compared to the original GFc-C35-3 / 15 peptide. Furthermore, the binding rate constant was ka = 4.6 × 10⁻⁶. 5 M- 1 S- 1 It is extremely fast; furthermore, the dissociation rate constant is kd = 2.5 × 10⁻⁻¹ 2 S- 1 .

[0400] The affinity (Kd value) of GFc-C35-3 / 15 (T5A, Y6W, T7H, N8R, N10E) is 257 nM, which is higher than that of the original GFc-C35-3 / 15 peptide. The dissociation reaction rate constant kd value is 1.4 × 10⁻⁻¹. 2 S- 1 The concentration of GFc-C35-3 / 15 (T5A, T7H, N8R, N10E) was reduced to half, which contributed to the increase in affinity. On the other hand, the binding rate constant ka was 5.4 × 10⁻⁶. 4 M- 1 S- 1 (Below 1 / 8), a significant decrease, therefore, the overall affinity of GFc-C35-3 / 15 (T5A, Y6W, T7H, N8R, N10E) is reduced. As mentioned above, the substitution of Y6W shows that it is an amino acid substitution that significantly affects both the ka and kd values.

[0401] The affinity of GFc-C35-3 / 15 (D3R, T5A, T7H, N8R, N10E, T15S) is 147 nM, which is higher than that of the original GFc-C35-3 / 15 peptide. However, the affinity is lower than that of GFc-C35-3 / 15 (T5A, T7H, N8R, N10E).

[0402] Through the above research, GFc-C35-3 / 15 (T5A, T7H, N8R, N10E) with an affinity approximately 2400 times that of the original peptide GFc-C35-3 / 15 was obtained. The peptide exhibiting the highest affinity will be referred to as C35A-3 / 15 below.

[0403] As shown in Table 5, the Kd values ​​of peptides with one or two residues deleted from each end of the GFc-C35 sequence (GFc-C35-2 / 15) (GFc-C35-3 / 15) decreased compared to GFc-C35, indicating that the residues at both ends (positions 1, 2, 16, and 17) facilitate binding. Therefore, the contribution of the two residues at both ends to affinity was evaluated. For C35A-3 / 15, which showed the highest affinity, the synthesis involved adding Glu at position 1. y Affinity analysis was performed by adding Pro at position 2, Phe at position 16, and His at position 17.

[0404] To confirm the effect of the addition of these four amino acid residues, a peptide (C35A(D3R,T15S)) with Gly added at position 1, Pro added at position 2, Phe added at position 16, and His added at position 17 was synthesized and affinity analysis was performed. Furthermore, C35A-3 / 15(D3R,T15S) has the same sequence as GFc-C35-3 / 15(D3R,T5A,T7H,N8R,N10E,T15S) in Table 6.

[0405] The results are shown in Table 7.

[0406] [Table 7]

[0407] Peptide Sequence Kd (μM) C35A-3 / 15 DCAYHRGELVWCT SEQ ID NO: 55 0.054 C35A GPDCAYHRGELVWCTFH SEQ ID NO: 56 0.009 C35A-3 / 15(D3R, T15S) RCAYHRGELVWCS SEQ ID NO; 57 0.147 C35A(D3R, T15S) GPRCAYHRGELVWCSFH SEQ ID NO: 58 0.042

[0408] When Gly, Pro, Phe, and His peptides (C35A) were inserted at both ends of the C35A-3 / 15 sequence, an increase in affinity (approximately 1 / 5 of the Kd value) was observed compared to the original sequence. Furthermore, even when the same amino acid peptides (C35A(D3R, T15S)) were inserted at both ends of the C35A-3 / 15 (D3R, T15S) sequence, an increase in affinity (approximately 1 / 3 of the Kd value) was also observed compared to the original sequence.

[0409] The introduction of two residues (positions 1, 2, 16, and 17) at both ends of the two peptides showed an increased affinity, indicating that the outer sequence of the Cys residues also contributes to affinity for human IgG. In other words, optimization of the outer sequence of the Cys residues showed that sequences with higher affinity could be obtained. Therefore, a library (Lib-D) was constructed in which the sequence inside the two Cys residues was fixed and the amino acids outside the Cys residues were randomized using a mixture of XYZ nucleotides. Figure 6 The sequence of peptides obtained through biopaneling is used to evaluate the characteristics of the amino acids that preferentially bind.

[0410] Specifically, a 5×10¹¹ pfu T7 phage library (X3CAYHRGELVWCX3) solution was added to the wells of a 96-well microplate (Nunc, Maxisorp) coated with RNase (derived from bovine pancreas, SIGMA) (4 μg / 400 μl / well) and blocked with 0.5% BSA, and allowed to react for 1 hour (adsorption step 1). Next, the supernatant was added to the wells of a 96-well microplate (Nunc, Maxisorp) coated with HSA (human serum albumin, SIGMA) (4 μg / 400 μl / well) and blocked with 0.5% BSA, and allowed to react for 1 hour (adsorption step 2). Then, the supernatant was transferred to the wells coated with human IgG1 (monoclonal, Chugai Pharmaceutical Co., Ltd.) (1 μg / 200 μl / well) and blocked with 0.5% BSA, and reacted for 1 hour (binding step). After removing the supernatant, the phage solution was washed three times with PBS containing 0.1% Tween (washing step). 200 μl of E. coli BLT5403 (Novagen) culture medium was added to infect the phage, and the mixture was incubated with 10 mL of E. coli culture medium at 37°C until proliferation and lysis (proliferation step). The phage was recovered from the lysed culture medium using a polyethylene glycol phage precipitation method according to standard procedures. The obtained phage was dissolved in PBS for the following panning. Seven rounds of panning, including the above-mentioned rounds, were performed to concentrate IgG-specific phage. In rounds 2–7, the number of washes was gradually increased, up to a maximum of 30. No adsorption step was performed in rounds 6–7. After the seventh panning, the phage was monocloned, and its binding specificity to human IgG was evaluated by ELISA. The peptide sequences of clones showing high binding activity to human IgG within the evaluated phage were analyzed. The results are shown in Table 8. [Table 8]

[0411] Clone Name Sequence 1-14 NDTCAYHRGELVECTYS SEQ ID NO: 59 1-15 SDSCAYHRGELVWCDGY SEQ ID NO: 60 1-16 VDSCAYHRGELVWCSNY SEQ ID NO: 61 1-23 SAECAYHRGELVWCSVF SEQ ID NO: 62 1-27 FNDCAYHRGELVWCSGY SEQ ID NO: 63 1-36 HETCAYHRGELVWCDHH SEQ ID NO: 64 1-38 ​ ​ 1-55 ​ ​ 2-1 ​ ​ 2-17 ​ ​ 2-18 ​ ​ 2-25 ​ ​ 2-33 ​ ​ 2-36 ​ ​ 2-38 ​ ​ 2-71 ​ ​ 2-72 ​ ​ 2-80 ​ ​

[0412] Based on the obtained peptide sequence, the frequency of amino acids found at each site is shown in the table. ​ .

[0413] At position 1, N-terminus 1, Ser is dominant, while Gly is the second most frequent amino acid in the original sequence (C35A). At position 2, Asp and Gly are dominant. At position 3, Ser residues are dominant, while Asp is the second most frequent amino acid found in the original sequence. At position 15, C-terminus 1, Ser frequency is significantly increased. At position 16, His and Gly frequencies are increased. At position 17, Tyr is dominant, with Tyr and Phe containing aromatic rings being dominant. These results show that by introducing these amino acids into the outer sequence of two Cys residues, peptides with higher affinity for human IgG can be designed.

[0414] Based on the above research, the effectiveness of using synthetic peptides to introduce amino acids that are found at high frequency at various sites was evaluated and verified.

[0415] Based on the synthetic peptide of C35A, peptides were synthesized by introducing amino acid mutations to replace the amino acids observed at high frequencies in the aforementioned phage library. Similar to the above, surface plasmon resonance (SPR) analysis was used to analyze the binding to human IgG, evaluating the contribution of each amino acid replacement to affinity. The results are shown in Table 9 below.

[0416] [Table 9]

[0417] ​ ​ ​ ​ SPDCAYHRGELVWCTFH SEQ ID NO: 100 0.0093 C35A(P2D) GDDCAYHRCELVWCTFH SEQ ID NO: 101 0.0133 C35A(D3S) GPSCAYHRGELVWCTFH SEQ ID NO: 102 0.0285 C35A(T15S) GPDCAYHRGELVWCSFH SEQ ID NO: 103 0.0144 C35A(F16H) GPDCAYHRGELVWCTHH SEQ ID NO: 104 0.0114 C35A(H17Y) GPDCAYHRGELVWCTFY SEQ ID NO: 105 0.012 C35A(C1S, H17Y) SPDCAYHRGELVWCTFY SEQ ID NO: 106 0.011 C35A(P2D, H17Y) [[ID= ​ 0.013

[0418] To confirm the usefulness of the obtained IgG-binding peptides, IgG was purified from human serum using a column immobilized with the IgG-binding peptides. The column immobilized with the IgG-binding peptides was prepared using the following method.

[0419] Add 1 mL of peptide dissolving solution (a 1.5 mM solution prepared by dissolving 4 mg of 4.0 mg of N-terminally PEGylated C35A-3 / 15 (NH2-PEG4-DCAYHRGELVWCT-NH2) in 1.1 mL of 0.05 M carbonate buffer (pH 8.3)) to a HiTrap NHS-activated HP column (1 mL, GE Healthcare) equilibrated with 5 mL of 1 mM HCl. Fix for 30 minutes at room temperature. Then wash with 1 mL of 1 M Tris, add 2 mL of 1 M Tris, and block for 30 minutes at room temperature. Wash three times with 6 mL of PBS, followed by one wash with 1 mL of PBS. Prepare a peptide immobilization column with an immobilization capacity of 1.2 μmol using the above procedure.

[0420] The obtained peptide immobilization column (1.2 μmol) was used in the Profinia protein purification system (BIO RAD). Human serum (1 mL) was diluted 5-fold, and the resulting PBS solution (5 mL) was applied to the peptide immobilization column. After washing the column with PBS, stepwise elution was performed with 0.1 M glycine hydrochloride (pH 2.5). Protein elution from the column was tracked using absorbance at 280 nm.

[0421] The results are shown in ​ .

[0422] The protein recovery was confirmed to be 10.4 mg by the absorbance of the elution fraction solution (fraction D, 5 mL).

[0423] about ​The elution fraction shown was used to confirm IgG in SDS-PAGE (reduction treatment) according to a known method. Specifically, elution fraction D was reduced with 2-mercaptoethanol and then electrophoresed on a 4–20% polyacrylamide gradient gel (Mini-PROTEAN TGX gel; BioRad), stained with Gelcode Blue Regent. The results showed clear bands around 25 kDa and 50 kDa for the light chain (L chain) and heavy chain (H chain) of IgG. ​ (5). Lanes 1, 2, 3, and 4 represent samples of molecular weight markers, human IgG products, HAS, and human serum.

[0424] Therefore, it is clear that columns immobilized with IgG-binding peptides can be used as affinity columns for the purification of human IgG.

[0425] Industrial availability

[0426] The present invention provides a peptide that can specifically or selectively bind to human IgG, thereby enabling its industrial use in the purification and analysis of IgG in the manufacture of IgG as an antibody drug.

[0427] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. A peptide, characterized in that, It contains an amino acid sequence of 13 to 17 amino acid residues as shown in Formula I below, and can bind to human IgG: (X 1-3 )-C-(X2)-H-R-G-(Xaa1)-L-V-W-C-(X 1-3 ) (I) In the formula, X represents any amino acid residue other than cysteine. C is a cysteine ​​residue. H stands for histidine residue. R represents an arginine residue. G stands for a glycine residue. Xaa1 is a glutamic acid residue or an asparagine residue. L represents a leucine residue. V represents a valine residue. And W is a tryptophan residue.

2. The peptide according to claim 1, characterized in that, It contains an amino acid sequence of 13 to 17 amino acid residues as shown in Formula II below, and can bind to human IgG: (X 1-3 )–C–(Q2)–(Q3)–HRG–(Qa1)–LVWC–(X 1-3 ) (II) In the formula, X represents any amino acid residue other than cysteine. C is a cysteine ​​residue. H stands for histidine residue. R represents an arginine residue. G stands for a glycine residue. Xaa1 is a glutamic acid residue or an asparagine residue. L represents a leucine residue. V represents a valine residue. W represents a tryptophan residue. Xaa2 is an alanine residue, a serine residue, or a threonine residue, and Xaa3 is a tyrosine residue or a tryptophan residue.

3. The peptide according to claim 1 or 2, characterized in that, It contains an amino acid sequence of 13 to 17 amino acid residues as shown in Formula III below, and can bind to human IgG: (X 1-3 )-CAYHRGELVWC-(X 1-3 ) (III) In the formula, X represents any amino acid residue other than cysteine. C is a cysteine ​​residue. A is an alanine residue. Y represents a tyrosine residue. H stands for histidine residue. R represents an arginine residue. G stands for a glycine residue. E represents a glutamic acid residue. L represents a leucine residue. V represents a valine residue. And W is a tryptophan residue.

4. The peptide according to any one of claims 1 to 3, wherein, When the number of amino acid residues is set to 17, the amino acid residues from the N-terminus, starting from the 1st to the 3rd and 15th to the 17th, are as follows: The first amino acid residue is either S, G, F, or absent; The second amino acid residue is either D, G, A, S, P, or absent; The third amino acid residue = S, D, T, N, E, or R; The 15th amino acid residue = S, T, or D; The 16th amino acid residue = H, G, Y, T, N, D, F or none; The 17th amino acid residue can be Y, F, H, M, or absent.

5. The peptide of claim 4, wherein the peptide comprises any of the following amino acid sequences 1) to 12): 1)DCAYHRGELVWCT (SEQ ID NO: 55) 2)GPDCAYHRGELVWCTFH (SEQ ID NO: 56) 3)RCAYHRGELVWCS (SEQ ID NO: 57) 4)GPRCAYHRGELVWCSFH (SEQ ID NO: 58) 5)SPDCAYHRGELVWCTFH (SEQ ID NO: 100) 6) GDDCAYHRGELVWCTFH (SEQ ID NO: 101) 7) GPSCAYHRGELVWCTFH (SEQ ID NO: 102) 8)GPDCAYHRGELVWCSFH (SEQ ID NO: 103) 9)GPDCAYHRGELVWCTHH (SEQ ID NO: 104) 10)GPDCAYHRGELVWCTFY (SEQ ID NO: 105) 11)SPDCAYHRGELVWCTFY (SEQ ID NO: 106) 12)SDDCAYHRGELVWCTFY (SEQ ID NO: 107).

6. The peptide according to claim 1 or 2, characterized in that, It contains an amino acid sequence of 13 amino acid residues as shown in Formula IV, and can bind to human IgG: DC-(Xaa2)-(Xaa3)-HRG-(Xaa1)-LVWCT (IV) In the formula, D stands for aspartic acid residue. C is a cysteine ​​residue. H stands for histidine residue. R represents an arginine residue. G stands for a glycine residue. Xaa1 is a glutamic acid residue or an asparagine residue. L represents a leucine residue. V represents a valine residue. W represents a tryptophan residue. T stands for a threonine residue. Xaa2 is an alanine residue or a threonine residue. Furthermore, Xaa3 is either a tyrosine residue or a tryptophan residue.

7. The peptide of claim 6, wherein the peptide comprises any of the amino acid sequences 1) to 4) below: 1)DCTYHRGNLVWCT (SEQ ID NO: 47) 2)DCAYHRGNLVWCT (SEQ ID NO: 48) 3)DCTYHRGELVWCT (SEQ ID NO: 50) 4) DCAWHRGELVWCT (SEQ ID NO: 53).

8. A peptide, characterized in that, It contains an amino acid sequence of 13 amino acid residues as shown in Formula V, and can bind to human IgG: DC-(Xaa1)-(Xaa2)-(Xaa3)-(Xaa4)-G-(Xaa5)-L-(Xaa6)-WCT (V) In the formula, D stands for aspartic acid residue. C is a cysteine ​​residue. G stands for a glycine residue. L represents a leucine residue. W represents a tryptophan residue. T stands for a threonine residue. Xaa1 is an alanine residue, a serine residue, or a threonine residue. Xaa2 is a tryptophan residue or a tyrosine residue. Xaa3 is a histidine residue, arginine residue, serine residue, or threonine residue. Xaa4 is an asparagine residue or an arginine residue. Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue, and Xaa6 is an isoleucine residue or a valine residue.

9. The peptide according to any one of claims 1 to 8, wherein, The peptide forms a disulfide bond between two cysteine ​​(C) residues.

10. The peptide according to any one of claims 1 to 9, wherein, The peptide is linked to a label.

11. A fusion protein comprising a protein linked to a peptide according to any one of claims 1 to 10.

12. An immobilized peptide, formed by combining the peptide of any one of claims 1 to 10 with a solid.

13. A nucleic acid encoding the peptide according to any one of claims 1 to 10.

14. A method for purifying IgG, the purification method comprising binding the peptide of any one of claims 1 to 10 or the immobilized peptide of claim 12 to IgG, and releasing the bound IgG to recover the IgG.

15. A method for detecting IgG, the method comprising binding IgG in a sample to the peptide of any one of claims 1 to 10 or the immobilized peptide of claim 12 and detecting the bound IgG.

16. A kit for the analysis or purification of human IgG, said kit containing at least one of the peptides of any one of claims 1 to 10 or the immobilized peptide of claim 12.

17. An IgG separation column comprising the immobilized peptide of claim 12.

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