Separating agent

Abstract

The object of the present invention is to provide a separating agent whose dynamic binding capacity (DBC) for a target substance does not decrease significantly even when subjected to continuous CIP treatment under alkaline pH conditions. This object is achieved by providing a separating agent comprising a carrier and a protein, wherein the protein is a predetermined protein, and the surface of the carrier is chemically bonded to the lysine residue in the protein.

Description

[Technical Field]

[0001] The present invention relates to a separating agent. [Background technology]

[0002] Various technologies have been developed to improve the performance of affinity chromatography columns. For example, immunoglobulin-binding proteins have been developed in which protein A has been modified with amino acid substitutions that reduce the number of lysine residues present on the protein surface in helices 1 and 2, which are the immunoglobulin-binding regions, as much as possible, while increasing the number of lysine residues present on helix 3 and the surrounding protein surface as much as possible. These modified proteins have been reported to have high immunoglobulin-binding activity and to be stable even under acidic and alkaline pH conditions, which normally cause protein function to be lost (Patent Document 1).

[0003] Meanwhile, a method is known in which antibody variable regions are displayed on the surface of phages as single-chain antibodies (single chain Fv, scFv) using phage display, and phages that bind to desired antigens are selected. By analyzing the genes of the selected phages, the DNA sequence encoding the single-chain antibody that binds to the antigen can be determined. Then, a separation agent for the antigen can be produced using the single-chain antibody that binds to the antigen. To use a single-chain antibody as a separation agent for an antigen, it is common to produce the agent by binding the single-chain antibody to a carrier for the separation agent (Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-252368 [Patent Document 2] International Publication No. 2017 / 082213 [Patent Document 3] International Publication No. 2017 / 082214 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have found that when a separating agent having a single-chain antibody bound to the surface of a carrier is continuously subjected to cleaning-in-place (CIP) treatment under alkaline pH conditions, the dynamic binding capacity (DBC) of the separating agent for the target substance significantly decreases, depending on the amino acid sequence contained in the single-chain antibody. The separating agent is used to separate a target substance from a mixture of two or more water-soluble substances. An object of the present invention is to provide a separating agent whose dynamic binding capacity (DBC) for a target substance does not significantly decrease even when subjected to continuous CIP treatment under alkaline pH conditions, and preferably, in addition, to provide a separating agent whose dynamic binding capacity (DBC) for a target substance is improved. [Means for solving the problem]

[0006] The present inventors have found that the above problems can be solved by employing the following configurations (1) and (2) as the separating agent. (1) When the single-chain antibody contained in the separating agent forms a three-dimensional structure, a lysine residue present in the vicinity of the target substance recognition site is substituted with an amino acid residue other than lysine. (2) When the single-chain antibody contained in the separating agent forms a three-dimensional structure, amino acid residues other than the lysine residues present far from the target substance recognition site are substituted with lysine residues. Specifically, it is as follows:

[0007] The present invention provides a separation agent comprising a carrier and a protein, The protein is A protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus, or A protein consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, arranged in order from the N-terminus, One or more lysine residues present in two or more of the regions represented by the following (a) to (j) are conserved and / or one or more amino acid residues other than lysine residues are substituted with lysine residues, In each of the regions represented by the following (e), (f), and (j), 1 to 12 amino acid residues including one lysine residue or 2 to 12 optionally consecutive lysine residues may be inserted or added, all lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues; a protein in which the cysteine ​​residue at position 96 (as designated by the IMGT numbering system) in the amino acid sequence represented by SEQ ID NO: 3 is substituted with an amino acid residue other than a cysteine ​​residue, This is a separation agent in which the surface of the carrier and the lysine residue in the protein are chemically bonded. (a) a region of positions 14 to 18, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 1 (b) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (c) a region of positions 72 to 75 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (d) a region of positions 92 to 96 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (g) a region of positions 14 to 18 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (h) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (i) a region of positions 82 to 86 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

[0008] Another aspect of the present invention is a method for separating a water-soluble substance to which the protein binds from a mixture of two or more water-soluble substances, using the separating agent.

[0009] Another aspect of the present invention is a protein consisting of, in order from the N-terminus, the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3; or A protein consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, arranged in order from the N-terminus, One or more lysine residues present in two or more of the regions represented by the following (a) to (j) are conserved and / or one or more amino acid residues other than lysine residues are substituted with lysine residues, In each of the regions represented by the following (e), (f), and (j), 1 to 12 amino acid residues including one lysine residue or 2 to 12 optionally consecutive lysine residues may be inserted or added, all lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues; This is a protein in which the cysteine ​​residue at position 96, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 3 is substituted with an amino acid residue other than cysteine. (a) a region of positions 14 to 18, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 1 (b) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (c) a region of positions 72 to 75 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (d) a region of positions 92 to 96 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (g) a region of positions 14 to 18 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (h) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (i) a region of positions 82 to 86 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3 [Effects of the Invention]

[0010] The present invention provides a separating agent whose dynamic binding capacity (DBC) for a target substance does not significantly decrease even when subjected to continuous CIP treatment under alkaline pH conditions. Preferably, the present invention also provides a separating agent whose dynamic binding capacity (DBC) for a target substance is improved. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the relationship between the amount of protein immobilized on an affinity chromatography column and 10% dynamic binding capacity (DBC) according to one embodiment of the present invention. [Figure 2] 1 is a graph showing the relationship between the amount of protein immobilized on an affinity chromatography column and 10% dynamic binding capacity (DBC) according to one embodiment of the present invention. [Figure 3] 1 is a graph showing the relative 10% dynamic binding amount (%) after each CIP treatment, when the 10% dynamic binding amount without CIP treatment is set to 100%, according to one embodiment of the present invention. [Figure 4-1] FIG. 1 shows the amino acid sequence shown in SEQ ID NO: 1 and an amino acid sequence substantially identical thereto, according to one embodiment of the present invention. [Figure 4-2] FIG. 1 shows the amino acid sequence shown in SEQ ID NO: 3 and an amino acid sequence substantially identical thereto, according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention includes an invention relating to a separating agent (first invention) and an invention relating to a method for separating a water-soluble substance to which the protein binds from a mixture of two or more water-soluble substances using the separating agent (second invention). In the art, single-chain antibodies are sometimes referred to as single-chain Fvs (scFvs) as a type of low-molecular-weight antibody. The single-chain antibodies described herein include rabbit-derived single-chain antibodies, but this is merely one example of a single-chain antibody derived from a rabbit, and the single-chain antibodies described herein are not limited to rabbit-derived single-chain antibodies.

[0013] <1. First Invention> A first aspect of the present invention is a separating agent comprising a carrier and a protein, The protein is A protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus, or A protein consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, arranged in order from the N-terminus, One or more lysine residues present in two or more of the regions represented by the following (a) to (j) are conserved and / or one or more amino acid residues other than lysine residues are substituted with lysine residues, In each of the regions represented by the following (e), (f), and (j), 1 to 12 amino acid residues including one lysine residue or 2 to 12 optionally consecutive lysine residues may be inserted or added, all lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues; a protein in which the cysteine ​​residue at position 96 (as designated by the IMGT numbering system) in the amino acid sequence represented by SEQ ID NO: 3 is substituted with an amino acid residue other than a cysteine ​​residue, This is a separation agent in which the surface of the carrier and the lysine residue in the protein are chemically bonded. (a) a region of positions 14 to 18, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 1 (b) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (c) a region of positions 72 to 75 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (d) a region of positions 92 to 96 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (g) a region of positions 14 to 18 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (h) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (i) a region of positions 82 to 86 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

[0014] [Carrier] The carrier used in the first aspect of the present invention is preferably a water-insoluble carrier. Examples of water-insoluble carriers include inorganic carriers such as glass beads and silica gel; organic carriers made of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; and polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran. Furthermore, composite carriers such as organic-organic and organic-inorganic carriers obtained by combining these are also preferred. Hydrophilic carriers are preferred because they exhibit relatively low nonspecific adsorption and provide good selectivity for the proteins used in the first aspect of the present invention. Here, the term "hydrophilic carrier" refers to a carrier whose contact angle with water when the carrier's constituent compounds are formed into a flat plate is 60° or less. Examples of such carriers include polysaccharides such as cellulose, chitosan, and dextran; polyvinyl alcohol; saponified ethylene-vinyl acetate copolymer; polyacrylamide; polyacrylic acid; polymethacrylic acid; polymethyl methacrylate; polyacrylic acid-grafted polyethylene; polyacrylamide-grafted polyethylene; and glass.

[0015] Examples of commercially available carriers include porous cellulose gels GCL2000 and GC700, Sephacryl S-1000, which is a covalently crosslinked mixture of allyl dextran and methylene bisacrylamide, Toyopearl, an acrylate-based carrier, Sepharose CL4B, an agarose-based crosslinked carrier, and Eupergit C250L, an epoxy-activated polymethacrylamide. However, the first aspect of the present invention is not limited to these carriers and activated carriers. Each of the above carriers may be used alone, or any two or more types may be mixed. Furthermore, the water-insoluble carrier used in the first aspect of the present invention preferably has a large surface area and has a large number of appropriately sized pores, i.e., is porous, in view of the purpose and method of use of the separating agent.

[0016] The carrier may be in any form, such as beads, fibers, or membranes (including hollow fibers), and any form can be selected. Beads are particularly preferred because of the ease of preparing carriers with a specific exclusion limit molecular weight. Beads with an average particle size of 10 to 2500 μm are easy to use, and a range of 25 to 800 μm is particularly preferred because the protein used in the first aspect of the present invention can be easily immobilized on the carrier surface via lysine residues. Furthermore, it is advantageous if the surface of the carrier has functional groups that can be used for chemical bonding with lysine residues in the protein used in the first aspect of the present invention. Representative examples of such functional groups include hydroxyl, amino, aldehyde, carboxyl, thiol, silanol, amide, epoxy, succinylimide, acid anhydride, and iodoacetyl groups.

[0017] [Chemical bond between lysine in protein and carrier surface] Methods for chemically bonding the protein used in the first aspect of the present invention to the carrier surface via a lysine residue in the protein (immobilization method) include chemical bonding via the ε-amino group of the lysine residue, such as covalent bonding to the carrier by conventional coupling methods. Examples of coupling methods include those commonly used to immobilize proteins or peptides on carriers. Examples include activating the carrier by reacting it with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, etc. (or introducing reactive functional groups onto the carrier surface) and then coupling with the lysine residue, or adding a condensation reagent such as carbodiimide or a reagent containing multiple functional groups in the molecule, such as glutaraldehyde, to a system containing the carrier and the lysine residue to cause condensation and crosslinking. However, it is more preferable to use a bonding method that prevents the lysine residue from easily detaching from the carrier during sterilization or use of the separating agent. Alternatively, a spacer molecule consisting of multiple atoms may be introduced between the lysine residue and the carrier, or the lysine residue may be directly immobilized on the carrier.

[0018] A specific example of a method for chemically bonding the protein used in the first aspect of the present invention to the carrier surface via lysine residues in the protein is an immobilization method using HiTrap NHS-activated HP Columns (GE Healthcare), as described in the Examples.

[0019] [protein] The protein used in the first aspect of the present invention is A protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus, or A protein consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, arranged in order from the N-terminus, One or more lysine residues present in two or more of the regions represented by the following (a) to (j) are conserved and / or one or more amino acid residues other than lysine residues are substituted with lysine residues, In each of the regions represented by the following (e), (f), and (j), 1 to 12 amino acid residues including one lysine residue or 2 to 12 optionally consecutive lysine residues may be inserted or added, all lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues; This is a protein in which the cysteine ​​residue at position 96, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 3 is substituted with an amino acid residue other than cysteine. (a) a region of positions 14 to 18, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 1 (b) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (c) a region of positions 72 to 75 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (d) a region of positions 92 to 96 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (g) a region of positions 14 to 18 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (h) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (i) a region of positions 82 to 86 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

[0020] The amino acid sequence represented by SEQ ID NO: 1 corresponds to the variable region (V) of the heavy chain (H chain) of the single-chain antibody R3-26 described in Patent Documents 2 and 3. H The amino acid sequence of the ATP domain. The amino acid sequence represented by SEQ ID NO: 3 corresponds to the variable region (V) of the light chain (L chain) in the single-chain antibody R3-26 described in Patent Documents 2 and 3. L The amino acid sequence of the ATP domain. The amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 16 are identical to the V amino acid sequence of the single-chain antibody R3-26 described in Patent Documents 2 and 3. H Domains and V L It is a linker sequence that connects the domains.

[0021] Patent Documents 2 and 3 indicate that the single-chain antibody R3-26 recognizes and binds to a human serum-derived IgG polyclonal antibody. Patent Documents 2 and 3 also indicate that the single-chain antibody R3-26 recognizes and binds to one or more antibodies selected from the group consisting of human serum-derived IgG1 polyclonal antibody, human serum-derived IgG2 polyclonal antibody, human serum-derived IgG3 polyclonal antibody, and human serum-derived IgG4 polyclonal antibody, which are its subtypes. The single-chain antibody R3-26 is a rabbit-derived single-chain antibody, but there are no particular limitations on the animal from which the single-chain antibody is derived. Examples of animals from which single-chain antibodies are derived include humans, rats, mice, rabbits, chickens, goats, sheep, cows, horses, dogs, cats, and monkeys.

[0022] In the protein used in the first aspect of the present invention, all of the lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues. The amino acid residues other than cysteine ​​residues are preferably arginine residues, serine residues, threonine residues, or glutamic acid residues. Furthermore, in the protein used in the first aspect of the present invention, the cysteine ​​residue at position 96 (IMGT numbering) in the amino acid sequence of SEQ ID NO: 3 is substituted with an amino acid residue other than cysteine, preferably an arginine residue, a serine residue, a threonine residue, or a glutamic acid residue.

[0023] Furthermore, the region (e) of the protein used in the first aspect of the present invention is, as described above, "a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1," but it may also be "a region of 1 to 5 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1." Furthermore, in the region (e), the "one to twelve amino acid residues including one lysine residue or two to twelve lysine residues, which may be consecutive, may be inserted or added" is preferably added to the C-terminal side of the first amino acid residue from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1. This is because it is advantageous for preserving the structure of the original protein if the lysine residue is located farther from amino acids that may be involved in forming the higher-order structure of the protein.

[0024] Furthermore, the region (f) of the protein used in the first aspect of the present invention is, as described above, "a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16," but it may also be "a region of 1 to 5 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16."

[0025] Furthermore, the region (j) of the protein used in the first aspect of the present invention is, as described above, "a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3," but it may also be "a region of 1 to 5 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3," or "a region of 1 to 3 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3." Furthermore, in the region (j), the "one or more amino acid residues other than lysine residues" in the "one or more amino acid residues other than lysine residues are substituted with lysine residues" is preferably an amino acid residue present in a region of 1 to 3 amino acid residues from the C-terminus in the amino acid sequence shown in SEQ ID NO: 3. Furthermore, in the region (j), the "1 to 12 amino acid residues including 1 lysine residue or 2 to 12 lysine residues, which may be consecutive, may be inserted or added" in the above "1 to 12 amino acid residues including 1 lysine residue or 2 to 12 lysine residues, which may be consecutive, may be inserted or added" is preferably inserted in a region of 1 to 3 amino acid residues from the C-terminus in the amino acid sequence shown in SEQ ID NO: 3, or added to the C-terminal side of the 1 amino acid residue from the C-terminus in the amino acid sequence shown in SEQ ID NO: 3.

[0026] The amino acid sequence shown in SEQ ID NO: 1 may be substantially equivalent to that sequence, as long as it can achieve the effects of the present invention. An amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence that is not represented by SEQ ID NO: 1 but can exhibit the effects of the present invention, and is V H When comparing amino acid sequences excluding the CDR regions of the domains, amino acid sequences that have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, and 95% or more are, in order of increasing preference. Examples of such amino acid sequences include SEQ ID NO: 21 (homology: 79.3%), SEQ ID NO: 22 (homology: 79.3%), SEQ ID NO: 23 (homology: 88.6%), SEQ ID NO: 24 (homology: 86.2%), SEQ ID NO: 25 (homology: 81.6%), SEQ ID NO: 26 (homology: 81.8%), SEQ ID NO: 27 (homology: 80.5%), and SEQ ID NO: 28 (homology: 81.6%). Furthermore, amino acid sequences that have a homology of preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more to each of these amino acid sequences are also included in amino acid sequences that are substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1.

[0027] Furthermore, the amino acid sequence shown in SEQ ID NO: 3 may be substantially equivalent to that sequence, as long as it can achieve the effects of the present invention. An amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 is an amino acid sequence that is not represented by SEQ ID NO: 3 but can exhibit the effects of the present invention, and is V L When comparing amino acid sequences excluding the CDR regions of the domains, amino acid sequences that have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, and 95% or more are, in order of increasing preference. Examples of such amino acid sequences include SEQ ID NO: 29 (homology: 78.0%), SEQ ID NO: 30 (homology: 89.0%), SEQ ID NO: 31 (homology: 86.4%), SEQ ID NO: 32 (homology: 81.3%), SEQ ID NO: 33 (homology: 87.9%), SEQ ID NO: 34 (homology: 85.7%), SEQ ID NO: 35 (homology: 82.4%), and SEQ ID NO: 36 (homology: 92.3%). Furthermore, amino acid sequences that have a homology of preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more to each of these amino acid sequences are also included in amino acid sequences that are substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3.

[0028] Furthermore, when the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 3 are assumed to be linked, the amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 has a V H The amino acid sequence of the CDR region of the domain, and V L The amino acid sequences may have a homology score calculated by BLOSUM62 of NCBI excluding the amino acid sequence of the CDR region of the domain of 500 or more, 550 or more, 600 or more, 650 or more, or 700 or more, in order of increasing preference. H The amino acid sequence of the CDR region of the domain, and V L The homology calculated excluding the amino acid sequence of the CDR region of the domain may be, in order of increasing preference, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0029] Such amino acid sequences include, for example: SEQ ID NO: 21 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 29 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 718, identity: 78.70%), SEQ ID NO: 22 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 30 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 762, identity: 84.30%), SEQ ID NO: 23 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 31 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 807, identity: 87.50%), SEQ ID NO: 24 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 32 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 737, identity: 80.90%), SEQ ID NO: 25 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 33 as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 763, identity: 84.80%), SEQ ID NO: 26 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 34 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 766, identity: 83.80%), SEQ ID NO: 27 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 35 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 740, identity: 81.50%), SEQ ID NO: 28 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1, and SEQ ID NO: 36 is an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3 (homology score: 744, identity: 87.10%). Furthermore, amino acid sequences that have a homology of preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with each of these amino acid sequences are also included in the amino acid sequences that are substantially equivalent to the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence that is substantially equivalent to the amino acid sequence represented by SEQ ID NO: 3, respectively.

[0030] The amino acid sequence shown in SEQ ID NO: 2 may be substantially equivalent to that sequence, as long as it can achieve the effects of the present invention. An amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence that is not the amino acid sequence represented by SEQ ID NO: 2 but can achieve the effects of the present invention, and is an amino acid sequence that has a homology of preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more to the amino acid sequence represented by SEQ ID NO: 2. This also applies to the amino acid sequence shown in SEQ ID NO:16.

[0031] Figure 4-1 shows the amino acid sequence shown in SEQ ID NO: 1 and a substantially identical amino acid sequence, and Figure 4-2 shows the amino acid sequence shown in SEQ ID NO: 3 and a substantially identical amino acid sequence. The clone numbers in each figure are the clone numbers of each single-chain antibody described in Patent Documents 2 and 3, and each sequence represents the V of each single-chain antibody. H Domains and V L The domain sequences are shown, along with the CDR and FR regions of each domain.

[0032] Furthermore, the amino acid sequence represented by SEQ ID NO: 1 may have one or more amino acids substituted, deleted, inserted and / or added, as long as the effects of the present invention can be achieved. "One to more" refers to, in order of increasing preference, 1 to 12, 1 to 10, 1 to 5, and 1 to 3 amino acids. Furthermore, the amino acid sequence represented by SEQ ID NO: 3 may have one or more amino acids substituted, deleted, inserted and / or added, as long as the effects of the present invention can be achieved. "One to more" refers to, in order of increasing preference, 1 to 12, 1 to 10, 1 to 5, and 1 to 3 amino acids. Furthermore, the amino acid sequence represented by SEQ ID NO: 2 may have one or more amino acids substituted, deleted, inserted and / or added, as long as the effects of the present invention can be achieved. "One or more" refers to, in order of increasing preference, 1 to 3 amino acids, 1 to 2 amino acids, and 1 amino acid. Furthermore, the amino acid sequence represented by SEQ ID NO: 16 may have one or more amino acids substituted, deleted, inserted and / or added, as long as the effects of the present invention can be achieved. "One or more" refers to, in order of increasing preference, 1 to 3 amino acids, 1 to 2 amino acids, and 1 amino acid.

[0033] The substitution, deletion, insertion and / or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 is a conservative mutation that maintains the normal function of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. A typical example of a conservative mutation is a conservative substitution, which is a mutual substitution between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, between Lys, Arg, and His when the substitution site is a basic amino acid, between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the substitution site is an amino acid with a hydroxyl group. Specific examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with L substitution of Leu with Ile, Met, Val or Phe; substitution of Lys with Asn, Glu, Gln, His or Arg; substitution of Met with Ile, Leu, Val or Phe; substitution of Phe with Trp, Tyr, Met, Ile or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, Ile or Leu. This also applies to the amino acid sequence represented by SEQ ID NO:2, the amino acid sequence represented by SEQ ID NO:16, and the amino acid sequence represented by SEQ ID NO:3.

[0034] Furthermore, as long as the effects of the present invention are achieved, the C-terminus of the protein used in the first aspect of the present invention may be tagged with a His tag, GST tag, FLAG tag, or the like. Furthermore, one or more linker amino acid sequences may be added to the N-terminus of these tags. The one or more amino acids are preferably 1 to 25, more preferably 1 to 15, and even more preferably 1 to 5 amino acids. Examples of linker sequences include AAALE (SEQ ID NO: 17), AAAGGGGSKIE (SEQ ID NO: 18), AAALE (SEQ ID NO: 19), and AAAGGGGSKKKKKIE (SEQ ID NO: 20).

[0035] The single-chain antibody R3-26 can be obtained by the methods described in Patent Documents 2 and 3, or by known genetic engineering techniques, protein engineering techniques, and the like. Furthermore, a protein consisting of an amino acid sequence substantially identical to that of the single-chain antibody R3-26 can be obtained by the methods described in Patent Documents 2 and 3, or by modifying the amino acid sequence of the single-chain antibody R3-26 by known genetic engineering techniques, protein engineering techniques, or the like. In either case, the method is not limited as long as it ultimately results in the single-chain antibody R3-26 or a protein consisting of an amino acid sequence substantially equivalent to the amino acid sequence of the single-chain antibody R3-26. Furthermore, the protein used in the first aspect of the present invention can be obtained by modifying the amino acid sequence of the single-chain antibody R3-26 or an amino acid sequence substantially equivalent to the amino acid sequence of the single-chain antibody R3-26 by known genetic engineering techniques, protein engineering techniques, etc., but the technique is not limited as long as it ultimately results in a protein consisting of the desired amino acid sequence.

[0036] [Dynamic Binding Capacity (DBC)] When conditions such as the amount of protein immobilized on the carrier and the flow rate are the same, the dynamic binding capacity (DBC) of the separation agent according to the first invention of the present invention is not smaller than, i.e., is larger than or equal to, the dynamic binding capacity (DBC) of a separation agent in which the single-chain antibody R3-26 is bound to the surface of the carrier via the original lysine residue in the amino acid sequence of the single-chain antibody. The dynamic binding capacity (DBC) of the separating agent according to the first aspect of the present invention, expressed as [10% DBC (mg / mL) at a flow rate of 1.0 mL / min] / [amount of protein immobilized on column (mg)], is preferably at least 1.00 times, more preferably at least 1.02 times, even more preferably at least 1.04 times, and even more preferably at least 1.05 times that of a separating agent in which the single-chain antibody R3-26 is bound to the surface of a carrier via an original lysine residue in the amino acid sequence of the single-chain antibody R3-26.

[0037] Furthermore, even when the separation agent according to the first aspect of the present invention is continuously subjected to CIP treatment under alkaline pH conditions, its dynamic binding capacity (DBC) is not smaller than, i.e., is larger than or the same as, the dynamic binding capacity (DBC) of a separation agent in which the single-chain antibody R3-26 is bound to the surface of a carrier via the original lysine residue in the amino acid sequence of the single-chain antibody. In this case, the dynamic binding capacity (DBC) of the separating agent according to the first aspect of the present invention is preferably at least 1.01 times, more preferably at least 1.05 times, and even more preferably at least 1.10 times that of a separating agent in which the single-chain antibody R3-26 is bound to the surface of a carrier via the original lysine residue in the amino acid sequence of the single-chain antibody R3-26, as expressed as [10% DBC (mg / mL) at a flow rate of 1.0 mL / min] / [amount of protein immobilized on the column (mg)], for example, after 10 cycles of the CIP treatment under alkaline pH conditions described in the Examples.

[0038] The dynamic binding capacity (DBC) of the separating agent according to the first aspect of the present invention can be measured by a known method, for example, by the method described in the Examples.

[0039] <2. Second Invention> The second aspect of the present invention is a method for separating a water-soluble substance to which the protein binds from a mixture of two or more water-soluble substances, using the separating agent according to the first aspect of the present invention. One of the two or more water-soluble substances is preferably a human serum-derived IgG polyclonal antibody, and more preferably one or more antibodies selected from the group consisting of its subtypes, i.e., human serum-derived IgG1 polyclonal antibody, human serum-derived IgG2 polyclonal antibody, human serum-derived IgG3 polyclonal antibody, and human serum-derived IgG4 polyclonal antibody. The method according to the second aspect of the present invention allows for sample analysis and separation of water-soluble substances to which the protein binds from the sample by the same procedures as in conventional affinity chromatography in which an antibody is immobilized on a carrier.

[0040] <3. The third invention> Another aspect of the present invention is A protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus, or A protein consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, arranged in order from the N-terminus, One or more lysine residues present in two or more of the regions represented by the following (a) to (j) are conserved and / or one or more amino acid residues other than lysine residues are substituted with lysine residues, In each of the regions represented by the following (e), (f), and (j), 1 to 12 amino acid residues including one lysine residue or 2 to 12 optionally consecutive lysine residues may be inserted or added, all lysine residues except for the conserved lysine residue, the substituted lysine residue, and the inserted or added lysine residue are substituted with amino acid residues other than cysteine ​​residues; This is a protein in which the cysteine ​​residue at position 96, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 3 is substituted with an amino acid residue other than cysteine. (a) a region of positions 14 to 18, as designated by the IMGT numbering system, in the amino acid sequence represented by SEQ ID NO: 1 (b) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (c) a region of positions 72 to 75 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (d) a region of positions 92 to 96 in the amino acid sequence represented by SEQ ID NO: 1, as designated by the IMGT numbering system; (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (g) a region of positions 14 to 18 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (h) a region of positions 45 to 50 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (i) a region of positions 82 to 86 in the amino acid sequence represented by SEQ ID NO: 3, as designated by the IMGT numbering system; (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

[0041] For details about the protein according to this embodiment, the explanation in the "Protein" section in the explanation of the first invention of the present invention is cited. Therefore, the protein according to this embodiment has the activity of binding to a human serum-derived IgG polyclonal antibody and, further, to one or more antibodies selected from the group consisting of its subtypes, i.e., human serum-derived IgG1 polyclonal antibody, human serum-derived IgG2 polyclonal antibody, human serum-derived IgG3 polyclonal antibody, and human serum-derived IgG4 polyclonal antibody. [Example]

[0042] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples as long as it does not deviate from the gist of the invention.

[0043] [Manufacturing Example 1] With reference to Patent Documents 2 and 3, a vector for expressing a protein (single-chain antibody R3-26 described in Patent Documents 2 and 3) was constructed from DNA (SEQ ID NO: 8) encoding the protein, which contains, in order from the N-terminus, the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3. The amino acid sequence of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus, is designated as SEQ ID NO: 4. Note that the amino acid sequence represented by SEQ ID NO: 4 does not include the amino acid sequence represented by SEQ ID NO: 12, which will be described later. The constructed single-chain antibody expression vector is expressed in a form in which a periplasmic transport signal (pelB Leader signal) sequence is fused to the N-terminus and AAALEHHHHHH (SEQ ID NO: 12) is fused to the C-terminus. After expression, the protein is transported to the periplasm, and the pelB Leader signal sequence is cleaved by signal peptidase.

[0044] E. coli Rosetta (DE3) was transformed with the constructed expression vector, and the transformed E. coli was cultured on an LB agar plate (50 mg / L ampicillin). The resulting single colony was cultured overnight in 10 mL of LB medium (50 mg / L ampicillin). The resulting culture was inoculated into 50 mL of Overnight Express TB medium (Merck Millipore) and cultured at 37°C and 200 rpm for 24 hours.

[0045] The resulting culture medium was centrifuged (10,000 rpm, 4°C, 15 minutes) to obtain the culture supernatant. This culture supernatant was filtered through a 0.45 μm pore size hydrophilic Durapore membrane (Merck Millipore), and the filtrate was applied to a HisTrap FF crude column (GE Healthcare) to capture the protein. The captured protein was eluted with 0.4 M imidazole. The protein concentration after elution was quantified by DC Protein assay (Biorad). Furthermore, the purity of the protein in the eluate was confirmed by SDS-PAGE. The solvent of the recovered protein solution was replaced with PBST, and the dissociation constant K was measured using a BiacoreX-100 as described below. D was measured.

[0046] [Manufacturing Example 2] The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 used in Production Example 1 was modified as follows. The modified protein is designated "R3-26 repCK+K" and its amino acid sequence is shown in SEQ ID NO: 5. Note that the amino acid sequence shown in SEQ ID NO: 5 does not include the amino acid sequence shown in SEQ ID NO: 13, which will be described later.

[0047] In the amino acid sequence represented by SEQ ID NO: 1, the lysine residue at position 14 designated by the IMGT numbering was substituted with a threonine residue, the lysine residue at position 48 designated by the IMGT numbering with a glutamic acid residue, and the lysine residues at positions 72, 80, and 90 designated by the IMGT numbering with arginine residues. In the amino acid sequence represented by SEQ ID NO: 2, no amino acid residues were substituted. In the amino acid sequence represented by SEQ ID NO:3, the lysine residue at position 22 (as designated by the IMGT numbering) was substituted with an asparagine residue, the lysine residue at position 51 (as designated by the IMGT numbering) with an arginine residue, the lysine residue at position 77 (as designated by the IMGT numbering) with a serine residue, and the cysteine ​​residue at position 96 (as designated by the IMGT numbering) with a serine residue. At the C-terminus of the amino acid sequence shown in SEQ ID NO: 3, AAAGGGGSKIEHHHHHH (SEQ ID NO: 13) was present in this order from the N-terminus to the C-terminus. The base sequence of the DNA encoding the amino acid sequence of R3-26 repCK+K is shown as SEQ ID NO:9.

[0048] [Manufacturing Example 3] The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 used in Production Example 1 was modified as follows. The modified protein is designated "R3-26 repCK+ori6K" and its amino acid sequence is shown in SEQ ID NO: 6. Note that the amino acid sequence shown in SEQ ID NO: 6 does not include the amino acid sequence shown in SEQ ID NO: 14, which will be described later.

[0049] In the amino acid sequence represented by SEQ ID NO: 1, the lysine residues at positions 14 and 48 designated by the IMGT numbering were preserved, the lysine residues at positions 72, 80, and 90 designated by the IMGT numbering were substituted with arginine residues, and the C-terminal serine residue was substituted with a lysine residue. In the amino acid sequence represented by SEQ ID NO: 2, no amino acid residues were substituted. the amino acid sequence represented by SEQ ID NO: 3, the alanine residue at position 14 designated by the IMGT numbering to a lysine residue, the lysine residue at position 22 designated by the IMGT numbering to an asparagine residue, the arginine residue at position 45 designated by the IMGT numbering to a lysine residue, the lysine residue at position 51 designated by the IMGT numbering to an arginine residue, the lysine residue at position 77 designated by the IMGT numbering to a serine residue, the cysteine ​​residue at position 96 designated by the IMGT numbering to a serine residue, and the third residue from the C-terminus Threonine The residue was replaced with a lysine residue. The C-terminus of the amino acid sequence represented by SEQ ID NO: 3 contains, in order from the N-terminus to the C-terminus: AAALEHHHHHH (SEQ ID NO: 14) was present. The base sequence of the DNA encoding the amino acid sequence of R3-26 repCK+ori6K is SEQ ID NO:10.

[0050] [Manufacturing Example 4] The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 used in Production Example 1 was modified as follows. The modified protein is designated "R3-26 repCK+K5" and its amino acid sequence is shown in SEQ ID NO: 7. Note that the amino acid sequence shown in SEQ ID NO: 7 does not include the amino acid sequence shown in SEQ ID NO: 15, which will be described later.

[0051] In the amino acid sequence represented by SEQ ID NO: 1, the lysine residue at position 14 designated by the IMGT numbering was substituted with a threonine residue, the lysine residue at position 48 designated by the IMGT numbering with a glutamic acid residue, and the lysine residues at positions 72, 80, and 90 designated by the IMGT numbering with arginine residues. In the amino acid sequence represented by SEQ ID NO: 2, no amino acid residues were substituted. In the amino acid sequence represented by SEQ ID NO:3, the lysine residue at position 22 (as designated by the IMGT numbering) was substituted with an asparagine residue, the lysine residue at position 51 (as designated by the IMGT numbering) with an arginine residue, the lysine residue at position 77 (as designated by the IMGT numbering) with a serine residue, and the cysteine ​​residue at position 96 (as designated by the IMGT numbering) with a serine residue. At the C-terminus of the amino acid sequence shown in SEQ ID NO: 3, AAAGGGGSKKKKKIEHHHHHH (SEQ ID NO: 15) was present in this order from the N-terminus to the C-terminus. The base sequence of the DNA encoding the amino acid sequence of R3-26 repCK+K5 is SEQ ID NO:11.

[0052] [Manufacturing Example 5] The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 used in Production Example 1 was modified as follows. The modified protein is designated "R3-26 repCK+ori6K+VH4KVL4K," and its amino acid sequence is shown in SEQ ID NO: 37. Note that the amino acid sequence shown in SEQ ID NO: 37 does not include the amino acid sequence shown in SEQ ID NO: 14, which will be described later.

[0053] In the amino acid sequence represented by SEQ ID NO: 1, the lysine residues at positions 14 and 48, as designated by the IMGT numbering, are preserved, the lysine residues at positions 72, 80, and 90, as designated by the IMGT numbering, are substituted with arginine residues, the serine residue at the C-terminus is substituted with a lysine residue, and four consecutive lysine residues are added to the C-terminus. In the amino acid sequence represented by SEQ ID NO: 2, no amino acid residues were substituted. the amino acid sequence represented by SEQ ID NO: 3, the alanine residue at position 14 designated by the IMGT numbering to a lysine residue, the lysine residue at position 22 designated by the IMGT numbering to an asparagine residue, the arginine residue at position 45 designated by the IMGT numbering to a lysine residue, the lysine residue at position 51 designated by the IMGT numbering to an arginine residue, the lysine residue at position 77 designated by the IMGT numbering to a serine residue, the cysteine ​​residue at position 96 designated by the IMGT numbering to a serine residue, and the third residue from the C-terminus Threonine The residue was replaced with a lysine residue, and four consecutive lysine residues were inserted between the second and third residues from the C-terminus. At the C-terminus of the amino acid sequence shown in SEQ ID NO: 3, AAALEHHHHHH (SEQ ID NO: 14) was present in this order from the N-terminus to the C-terminus. The base sequence of the DNA encoding the amino acid sequence of R3-26 repCK+ori6K+VH4KVL4K is shown as SEQ ID NO:38.

[0054] The dissociation constant K D The measurement was carried out under the following conditions. Sensor Chip: human IgG1-coupled CM5 Running buffer: PBST Binding time: 180 seconds Dissociation time: 600-800 seconds Elution: 10mM Glycine, pH 1.5 Mode: Single cycle kinetics mode

[0055] Table 1 shows the yields and dissociation constants K of the proteins obtained in Production Examples 1 to 3 and 5. D The results of the measurements are summarized below. Compared with the protein obtained in Production Example 1, no decrease in production amount was observed for any of the proteins obtained in Production Examples 2 and 3, and even in Production Example 5, the dissociation constant K Dwas of the same order or less. From the above results, it is clear that the modification of the amino acid sequence affects the productivity and dissociation constant K D It was found that this did not result in a significant decrease in the binding ability shown in

[0056] [Table 1]

[0057] [Reference example 1-1] (Preparation of single chain antibodies) The same procedure as in Preparation Example 1 was followed, and the solvent of the protein solution recovered by ultrafiltration was replaced with 0.2 M sodium bicarbonate buffer (pH 8.3) containing 0.5 M NaCl.

[0058] (Immobilization of proteins onto carriers) The purified protein was loaded onto a 1 mL HiTrap NHS-activated HP column (GE Healthcare) and immobilized via the amino group of the lysine residue contained in the protein. Unreacted NHS ester was blocked by adding tris(hydroxymethyl)aminomethane. The amount of the protein immobilized on the column was 4.0 mg.

[0059] (Measurement of dynamic binding amount) The column with the immobilized protein was placed in an AKTA Purifier UPC 10 (GH Healthcare) chromatography system and equilibrated with PBS. A 1 mg / mL human serum-derived IgG polyclonal antibody (WAKO) was continuously supplied to the column at a flow rate of 1 mL / min to obtain a breakthrough curve. The 10% dynamic binding capacity (DBC) was calculated from the elution volume at the 10% breakthrough point of the resulting breakthrough curve.

[0060] [Reference example 1-2] The procedure was the same as in Reference Example 1-1, except that the amount of protein immobilized on the column was 34.4 mg.

[0061] [Comparative Example 1] The procedure was the same as in Reference Example 1-1, except that the protein "R3-26 repCK+K" produced in Production Example 2 was used as the protein.

[0062] [Example 1] The same procedures as in Reference Example 1-1 were carried out except that the protein used was the protein "R3-26 repCK+ori6K" produced in Production Example 3, and the immobilized amount was 33.6 mg.

[0063] [result] The results are summarized in Table 2. The results for Reference Examples in the table are the average values ​​of the results for Reference Examples 1-1 and 1-2. When Example 1 was used, the DBC value per amount of immobilized protein was increased compared to when the Reference Example was used. On the other hand, when Comparative Example 1 was used, the DBC value per amount of immobilized protein was smaller than when the Reference Example was used. The above results indicate that the capture efficiency of target substances on the carrier can be improved by substituting lysine residues present near the target substance recognition site when the protein contained in the separation agent forms a three-dimensional structure with amino acid residues other than lysine residues, and by substituting amino acid residues other than lysine residues present far from the target substance recognition site with lysine residues. Furthermore, we found that improving the capture efficiency of target substances cannot be achieved by simply adding a linker amino acid sequence containing one lysine residue to the C-terminus of the protein; it is necessary to select an appropriate number and position of lysine residues.

[0064] [Table 2]

[0065] [Reference example 2] The procedure was the same as in Reference Example 1-1, except that the amount of protein immobilized on the column was changed over a wide range.

[0066] [Example 2] The procedure was the same as in Reference Example 2, except that the amount of the protein "R3-26 repCK+ori6K" produced in Production Example 3 immobilized on the column was changed over a wide range.

[0067] [result] The results are shown in Figure 1. The dashed line is an approximation of the resulting plot. From this result, it was found that Example 2 had a higher target substance capture efficiency than Reference Example 2 over a wide range of immobilization amounts.

[0068] [Reference example 3] The procedure was the same as in Reference Example 1-1, except that the amount of protein immobilized on the column was varied over a wide range, the AKTA pure 25 M1 chromatography system (GH Healthcare) was used for measuring dynamic binding capacity, and γ-Globulin from Human Blood (Sigma-Aldrich) was used as the antibody sample.

[0069] [Example 3] The procedure was the same as in Reference Example 3, except that the protein "R3-26 repCK+ori6K+VH4KVL4K" produced in Production Example 5 was used.

[0070] [result] The results are shown in Figure 2. The dashed line is an approximation of the resulting plot. From this result, it was found that Example 3 had a higher target substance capture efficiency than Reference Example 3 over a wide range of immobilization amounts.

[0071] (Evaluation of stability under alkaline pH conditions) [Reference example 4] The procedure was the same as in Reference Example 1-1, except that the amount of protein immobilized on the column was 8.5 mg. A 0.1 N NaOH solution was supplied to the column at 1 mL / min for 10 minutes, followed by equilibration with PBS (CIP treatment). The 10% dynamic binding capacity was measured using the column after the CIP treatment a predetermined number of times.

[0072] [Example 4] The same procedure as in Reference Example 4 was carried out except that the protein used was the protein "R3-26 repCK+ori6K" produced in Production Example 3, and the amount immobilized was changed to 9.8 mg.

[0073] Comparative Example 3 The protein used was the protein "R3-26 repCK+K5" produced in Production Example 4, and the immobilized amount was 9.9 mg. Except for this, a column was produced in the same manner as in Reference Example 1-1, and the same procedure as in Reference Example 4 was used.

[0074] [Example 5] The same procedures as in Reference Example 4 were performed, except that the protein used was the protein "R3-26 repCK+ori6K+VH4KVL4K" produced in Production Example 5, and the immobilization amount was changed to 15.7 mg, the chromatography system AKTA pure 25 M1 (GH Healthcare) was used for measuring dynamic binding capacity, and γ-Globulin from Human Blood (Sigma-Aldrich) was used as the antibody sample.

[0075] [result] FIG. 3 shows the relative 10% dynamic binding amount (%) after each CIP treatment, when the 10% dynamic binding amount without CIP treatment was set to 100%. In Reference Example 4, after 10 cycles of CIP treatment, the relative 10% dynamic binding capacity (%) was lower than 80%, whereas in Examples 4 and 5, after 10 cycles of CIP treatment, the relative 10% dynamic binding capacity (%) was higher than 80%. This indicates that by substituting lysine residues present near the target substance recognition site with amino acid residues other than lysine residues when the protein contained in the separating agent forms a three-dimensional structure, and by substituting amino acid residues other than lysine residues present far from the target substance recognition site with lysine residues, the dynamic binding capacity (DBC) did not decrease significantly even when CIP treatment was continued under alkaline pH conditions, and alkaline resistance was improved. On the other hand, the relative 10% dynamic binding amount (%) after 10 cycles of CIP treatment in Comparative Example 3 was significantly smaller than the relative 10% dynamic binding amount (%) after 10 cycles of CIP treatment in Reference Example 4, indicating a decrease in alkali resistance. These results indicate that in order to improve alkaline resistance, it is necessary not only to remove lysine residues from the vicinity of the target substance recognition site when the protein contained in the separation agent forms a three-dimensional structure, but also to place an appropriate number of lysine residues at an appropriate position distant from the target substance recognition site.

Claims

1. A separation agent comprising a carrier and a protein, the protein is a single chain antibody (scFv), The scFv is An scFv consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus; or An scFv consisting of, in order from the N-terminus, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, In the amino acid sequence represented by SEQ ID NO: 1, the lysine residues at positions 14 and 48 as designated by the IMGT numbering are conserved, the lysine residues at positions 72, 80, and 90 as designated by the IMGT numbering are substituted with arginine residues, and the serine residue at the C-terminus is substituted with a lysine residue; and In the amino acid sequence represented by SEQ ID NO: 3, the alanine residue at position 14 (as designated by the IMGT numbering), the arginine residue at position 45 (as designated by the IMGT numbering), and the threonine residue at the third residue from the C-terminus are substituted with lysine residues, and the lysine residue at position 22 (as designated by the IMGT numbering) is substituted with an asparagine residue, the lysine residue at position 51 (as designated by the IMGT numbering) is substituted with an arginine residue, the lysine residue at position 77 (as designated by the IMGT numbering) is substituted with a serine residue, and the cysteine ​​residue at position 96 (as designated by the IMGT numbering) is substituted with a serine residue, The scFv may have one or more and 12 or less amino acid residues inserted or added in each of the regions represented by the following (e), (f), and (j), including one lysine residue or two or more and 12 or less lysine residues which may be consecutive: A separation agent in which the surface of the carrier and the lysine residue in the protein are chemically bonded. (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

2. A method for separating a water-soluble substance to which said protein binds from a mixture of two or more water-soluble substances, using the separating agent according to claim 1.

3. A single-chain antibody (scFv) consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, arranged in order from the N-terminus; or A single-chain antibody (scFv) consisting of, in order from the N-terminus, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 1, In the amino acid sequence represented by SEQ ID NO: 1, the lysine residues at positions 14 and 48 as designated by the IMGT numbering are conserved, the lysine residues at positions 72, 80, and 90 as designated by the IMGT numbering are substituted with arginine residues, and the serine residue at the C-terminus is substituted with a lysine residue; and In the amino acid sequence represented by SEQ ID NO: 3, the alanine residue at position 14 (as designated by the IMGT numbering), the arginine residue at position 45 (as designated by the IMGT numbering), and the threonine residue at the third residue from the C-terminus are substituted with lysine residues, and the lysine residue at position 22 (as designated by the IMGT numbering) is substituted with an asparagine residue, the lysine residue at position 51 (as designated by the IMGT numbering) is substituted with an arginine residue, the lysine residue at position 77 (as designated by the IMGT numbering) is substituted with a serine residue, and the cysteine ​​residue at position 96 (as designated by the IMGT numbering) is substituted with a serine residue, An scFv in which, in each of the regions represented by (e), (f), and (j) below, 1 to 12 amino acid residues, including 1 lysine residue or 2 to 12 lysine residues which may be consecutive, may be inserted or added. (e) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (f) a region of 1 to 12 amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 or 16 (j) a region of 1 to 12 amino acid residues from the C-terminus in the amino acid sequence represented by SEQ ID NO: 3

Images