Immunoglobulin-binding proteins

Abstract

Disclosed is a protein which comprises an amino acid sequence derived from an immunoglobulin-binding domain of a protein L of a bacterium belonging to the genus Vincura, said amino acid sequence having at least one or more amino acid substitutions selected from the group consisting of (1) to (5) below. The protein has immunoglobulin binding activity: (1) an amino acid residue equivalent to tyrosine at position 42 of SEQ ID NO: 1 is substituted with histidine; (2) an amino acid residue equivalent to the lysine at position 22 of SEQ ID NO: 1 is substituted with glutamic acid; (3) an amino acid residue corresponding to the lysine at position 38 in SEQ ID NO: 1 is substituted with proline or aspartic acid; (4) an amino acid residue equivalent to tyrosine at position 42 in SEQ ID NO: 1 is substituted with tryptophan or phenylalanine; (5) The amino acid residue equivalent to the asparagine at position 44 in SEQ ID NO: 1 is substituted with proline.

Description

Technical Field

[0001] This invention relates to a protein that specifically binds to immunoglobulins. More specifically, this invention relates to an immunoglobulin-binding protein with enhanced binding selectivity to immunoglobulins belonging to a specific light chain subgroup. Background Technology

[0002] Antibody drugs utilize antibodies (immunoglobulins), molecules that perform immune functions in the body. Antibody drugs bind to target molecules with high specificity and affinity due to the diversity of their variable regions. Therefore, antibody drugs have fewer side effects, and in recent years, their indications have become increasingly broad, leading to a rapidly expanding market.

[0003] As a novel form of antibody used in antibody drugs, bispecific antibodies capable of binding to two different antigens simultaneously can be cited. For example, bispecific antibodies that recognize cancer cell antigens and effector cell antigens are expected to enhance antitumor activity by effectively binding to these cells. Examples of bispecific antibodies include trifunctional antibodies composed of an Fc region and two Fab regions capable of binding to different antigens respectively, F(ab')2 antibodies composed of adaptor peptides or the like linking the two Fab regions, single-chain Fv (scFv) antibodies composed of adaptor peptides or the like linking only the variable regions of the two Fab regions, or bispecific T cell-inducing (BiTE) antibodies (Non-Patent Literature 1).

[0004] The manufacture of antibody drugs includes culturing and purification processes. In the culturing process, efforts are underway to modify antibody-producing cells or optimize culture conditions to improve productivity. In the purification process, crude purification employs affinity chromatography to specifically recognize antibody molecules, followed by intermediate purification, final purification, and virus removal before formulation.

[0005] As ligand proteins used in affinity chromatography carriers, protein A, derived from Staphylococcus bacteria, is often used. This protein has the property of binding to antibodies (immunoglobulins). However, protein A specifically binds to the Fc region of antibodies. Therefore, it cannot be used to purify antibodies that do not have an Fc region, such as F(ab')2, scFv, and BiTE antibodies. On the other hand, protein L, derived from Fingoldii bacteria, is a protein that binds to the κ light chain of immunoglobulins. Therefore, by using protein L as a ligand protein, it is possible to purify antibodies that do not have an Fc region, which cannot be purified by protein A.

[0006] When antibodies are formed from two light chains and two heavy chains, there are a total of ten possible combinations of light and heavy chains. Therefore, when manufacturing bispecific antibodies, it is necessary to select bispecific antibodies composed of the correct combinations from these combinations (Non-Patent Document 1). As methods to prevent incorrect combinations, the Knob-into-Holes method (Non-Patent Document 2), the Cross-mab method (Non-Patent Document 3), and the use of common light chains (Non-Patent Document 3) can be cited. However, any of these methods requires modification of the antibody molecule, thus narrowing the scope of molecular design and raising concerns about decreased expression levels or performance associated with the modifications.

[0007] On the other hand, as a method for selecting the correct combination of light chains without modifying the antibody molecule, one example is to selectively select heterogeneous aggregates by designating one of the two light chains as the κ chain and the other as the λ chain, taking advantage of the difference in affinity with protein L. However, in general, the λ chain has poor stability and solubility compared to the κ chain, especially the κ chains belonging to subgroups 1 and 3 (Non-Patent Literature 5), and therefore may impair the stability of the bispecific antibody.

[0008] As mentioned above, the κ chains belonging to subgroups 1 and 3 exhibit excellent stability and solubility. Therefore, bispecific antibodies with one light chain belonging to subgroup 1 and the other to subgroup 3 could be expected to have improved stability. However, no protein L has been found that can selectively bind to immunoglobulins containing κ light chains belonging to specific subgroups, making it difficult to select the bispecific antibody using protein L. Existing technical documents Non-patent literature

[0009] Non-patent literature 1: Ulrich Brinkmann et al., MAbs., 2017; 9: 182-212. Non-patent literature 2: JB Ridgway et al., Protein Eng., 1996; 9: 617-621. Non-patent literature 3: Christian Klein et al., MAbs., 2016; 8: 1010-1020. Non-patent literature 4: A. Margaret Merchant et al., Nature Biotechnology, 1998; 16: 677-681 Non-patent literature 5: Dae Young Kim et al., MAbs., 2014; 6: 219-235. Summary of the Invention The problem the invention aims to solve

[0010] The objective of this invention is to provide an immunoglobulin-binding protein with enhanced binding selectivity to immunoglobulins having light chains belonging to a specific subgroup, and an adsorbent for immobilizing the protein. Solution for solving the problem

[0011] In order to solve the aforementioned problem, the inventors conducted in-depth research and found that by replacing the amino acid residues related to the selection of the light chain subgroup in the immunoglobulin binding domain of a protein L (FpL) from a specific Fingold bacteria with other specific amino acid residues, the binding selectivity for κ light chain subgroup 1 was improved.

[0012] That is, the present invention includes, for example, the methods described in [1] to

[14] below.

[0013] [1] A protein comprising an amino acid sequence of an immunoglobulin-binding domain of protein L derived from Fingoldii bacteria, wherein the amino acid sequence has at least one or more amino acid substitutions selected from (1) to (5) below, and the protein has immunoglobulin-binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline. [2] The protein according to [1] is the protein described in any one of (a) to (c) below: (a) A protein comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence has at least one or more amino acid substitutions selected from (1) to (5) below, and the protein has immunoglobulin binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline; (b) A protein comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence comprises at least one amino acid substitution selected from (1) to (5), and in addition to (1) to (5), it comprises substitution, deletion, insertion, and / or addition of one or more amino acid residues at one or more positions, and the protein has immunoglobulin binding activity. (c) A protein comprising an amino acid sequence having more than 70% homology with the amino acid sequence or a portion thereof described in sequence number 1, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5), and the protein has immunoglobulin binding activity. [3] The protein according to [1] or [2] further comprises at least one amino acid substitution selected from (6) to (11) of the following: (6) The amino acid residue equivalent to asparagine at position 50 of sequence number 1 is replaced with tyrosine. (7) The amino acid residue equivalent to glutamic acid at position 4 of sequence number 1 is replaced with glycine. (8) The amino acid residue equivalent to lysine at position 7 of sequence number 1 is replaced with alanine. (9) The amino acid residue equivalent to glutamic acid at position 52 of sequence number 1 is replaced with leucine. (10) The amino acid residue equivalent to tyrosine at position 53 of sequence number 1 is replaced with phenylalanine. (11) The amino acid residue equivalent to alanine at position 69 of sequence number 1 is replaced with valine. [4] The protein according to [1] or [2] further comprises at least the following amino acid substitutions (6) and one or more amino acid substitutions selected from (7) to (11): (6) The amino acid residue equivalent to asparagine at position 50 of sequence number 1 is replaced with tyrosine. (7) The amino acid residue equivalent to glutamic acid at position 4 of sequence number 1 is replaced with glycine. (8) The amino acid residue equivalent to lysine at position 7 of sequence number 1 is replaced with alanine. (9) The amino acid residue equivalent to glutamic acid at position 52 of sequence number 1 is replaced with leucine. (10) The amino acid residue equivalent to tyrosine at position 53 of sequence number 1 is replaced with phenylalanine. (11) The amino acid residue equivalent to alanine at position 69 of sequence number 1 is replaced with valine. [5] The protein according to any one of [1] to [4] further comprises at least the following (12) amino acid substitutions: (12) The amino acid residue equivalent to lysine at position 29 of sequence number 1 is replaced with isoleucine. [6] The protein according to [3] or [4] is any one of the following proteins described in (d) to (f): (d) A protein comprising the amino acid sequence described in sequence number 70, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5) below, and the protein has immunoglobulin binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline; (e) A protein comprising the amino acid sequence described in sequence number 70, wherein the amino acid sequence comprises at least one amino acid substitution selected from (1) to (5), and in addition to (1) to (5), it comprises substitution, deletion, insertion, and / or addition of one or more amino acid residues at one or more positions, and the protein has immunoglobulin binding activity. (f) A protein comprising an amino acid sequence having more than 70% homology with the amino acid sequence or a portion thereof described in sequence number 70, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5), and the protein has immunoglobulin binding activity. [7] A polynucleotide that encodes the protein described in any one of [1] to [6]. [8] An expression vector comprising the polynucleotide described in [7]. [9] An expression vector comprising a gene recombination host, said gene recombination host comprising either (A) or (B): (A) A polynucleotide that encodes the protein described in any one of [1] to [6]; (B) A polynucleotide that encodes the protein described in any one of [1] to [6].

[10] The host of the gene recombination according to [9] is Escherichia coli.

[11] A method for manufacturing a protein having immunoglobulin binding activity, comprising: a step of culturing a gene recombinant host containing a polynucleotide encoding the protein described in any one of [1] to [6] or a gene recombinant host containing an expression vector containing the polynucleotide and expressing the protein thereon; and a step of recovering the expressed protein.

[12] The host of the manufacturing method described in

[11] is Escherichia coli.

[13] An immunoglobulin adsorbent comprising an insoluble carrier and a protein of any one of [1] to [6] immobilized on the insoluble carrier.

[14] A method for separating immunoglobulins contained in solution, comprising: adding a solution containing immunoglobulins to a column filled with the adsorbent described in

[13] and adsorbing the immunoglobulins onto the adsorbent; and eluting the immunoglobulins adsorbed onto the adsorbent. Detailed Implementation

[0014] The present invention will now be described in detail.

[0015] The proteins of this invention are specific immunoglobulin-binding proteins. In this specification, "immunoglobulin-binding protein" refers to a protein that binds to immunoglobulins. That is, the proteins of this invention bind to specific immunoglobulins. Specifically, the proteins of this invention can be proteins that bind to the κ light chain of immunoglobulins. That is, specifically, the proteins of this invention can bind to immunoglobulins containing the κ light chain. More specifically, the proteins of this invention can be proteins that bind to the Vκ1 of immunoglobulins. That is, more specifically, the proteins of this invention can bind to immunoglobulins containing Vκ1. "Vκ1" refers to κ light chain subgroup 1, in other words, a light chain belonging to subgroup 1. In this specification, the binding to immunoglobulins is also referred to as "immunoglobulin binding activity" or "antibody binding activity." Immunoglobulin binding activity can be determined, for example, by an ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA method can be performed, for example, under the conditions described in the examples.

[0016] The protein of the present invention is a protein comprising an amino acid sequence of an immunoglobulin-binding domain of protein L (also referred to as "FpL") derived from Fingoldii bacteria, wherein an amino acid substitution is present at a specific position in this amino acid sequence. Hereinafter, in this specification, the amino acid sequence of the immunoglobulin-binding domain of FpL without the specific amino acid substitution is referred to as an "unmodified amino acid sequence," and the amino acid sequence of the immunoglobulin-binding domain of FpL with the specific amino acid substitution is referred to as a "modified amino acid sequence." That is, the modified amino acid sequence can be the same as the unmodified amino acid sequence except for the specific amino acid substitution. Furthermore, according to one embodiment of the present invention, the protein may, for example, be a protein comprising an amino acid sequence identical to the unmodified amino acid sequence except for the specific amino acid substitution. Additionally, according to one embodiment of the present invention, the protein may, for example, be a protein comprising a modified amino acid sequence. The unmodified amino acid sequence may or may not be a naturally occurring amino acid sequence. The unmodified amino acid sequence may, for example, be modified in a manner that has desired properties. The unmodified amino acid sequence may, for example, have amino acid substitutions other than the specific amino acid substitution.

[0017] Regarding Fingoldella bacteria that are sources of FpL, Finegoldiamagna is an example. As for the immunoglobulin-binding domain of protein L derived from Finegoldiamagna, the following can be cited: Domain B1 (amino acid residues from position 104 to 173 in GenBank No. AAA25612), Domain B2 (amino acid residues from position 176 to 245 of GenBank No. AAA25612), Domain B3 (amino acid residues from position 248 to 317 of GenBank No. AAA25612), Domain B4 (amino acid residues 320 to 389 of GenBank No. AAA25612), Domain B5 (amino acid residues from position 393 to 462 in GenBank No. AAA25612), Domain C1 (amino acid residues from position 249 to 317 in sequence number 106 (GenBank No. AAA67503)), Domain C2 (amino acid residues from position 320 to position 389 of sequence number 106), Domain C3 (amino acid residues 394 to 463 of sequence number 106, sequence number 1), and And the C4 domain (amino acid residues from position 468 to position 537 of sequence number 106).

[0018] In this specification, with regard to the unmodified amino acid sequence, (i) can be the complete amino acid sequence of the immunoglobulin-binding domain of the FpL described above. (ii) It can also be a partial amino acid sequence of the domain, as long as it has binding activity to immunoglobulins.

[0019] Furthermore, regarding (ii), the immunoglobulin-binding domain of FpL consists of four β-sheets and an α-helix, a loop connecting them, and an N-terminal loop region. However, amino acid residues in regions unrelated to immunoglobulin binding, such as the N-terminal loop region, may be missing. As a specific example, it is known that even with the absence of amino acid residues from position 1 to position 9 of the glutamic acid in domain C3 (Sequence No. 1) of FpL, which corresponds to the N-terminal loop region, it still possesses immunoglobulin-binding ability (Housden N.G. et al. Biochemical Society Transaction, 2003; 31: 716-718). That is, it can be considered that the partial amino acid sequence in (ii) only needs to contain at least the amino acid sequence of the immunoglobulin-binding site. Specifically, examples of partial amino acid sequences in (ii) that contain the amino acid sequence of the immunoglobulin-binding site can be cited.

[0020] Specifically, as a modified amino acid sequence in this specification, examples include amino acid sequences such as the amino acid sequence described in Serial No. 1, which have amino acid substitutions at specific positions in the amino acid sequence of the immunoglobulin-binding domain described above. That is, according to one embodiment of the present invention, as a protein, specifically, a protein can be described as comprising the amino acid sequence of the immunoglobulin-binding domain described above, such as the amino acid sequence described in Serial No. 1, wherein an amino acid substitution is present at a specific position in this amino acid sequence. In other words, according to one embodiment of the present invention, a protein may, for example, be a protein comprising an amino acid sequence identical to the amino acid sequence of the immunoglobulin-binding domain described above, such as the amino acid sequence described in Serial No. 1, except for the amino acid substitution at the specific position.

[0021] In this specification, the term "protein containing an amino acid sequence" is also used to refer to "a protein containing amino acid residues composed of an amino acid sequence." Additionally, the term "a protein or amino acid sequence having amino acid substitutions" is also used to refer to "an amino acid substitution is produced in a protein or amino acid sequence." Furthermore, the amino acids constituting a protein or amino acid sequence are referred to as "amino acid residues."

[0022] According to one embodiment of the present invention, the protein can be an immunoglobulin-binding protein with increased binding selectivity for Vκ1. Specifically, according to one embodiment of the present invention, the protein can be an immunoglobulin-binding protein with increased binding selectivity for Vκ1 compared to immunoglobulin-binding proteins without modified amino acid sequences (e.g., composed of unmodified amino acid sequences). Specifically, according to one embodiment of the present invention, the protein can be an immunoglobulin-binding protein with increased binding selectivity for Vκ1 due to amino acid substitution at the specific position. In other words, according to one embodiment of the present invention, the protein can be an immunoglobulin-binding protein with increased binding selectivity for Vκ1 through amino acid substitution at the specific position. Furthermore, in other words, the amino acid substitution at the specific position can be an amino acid substitution that increases the binding selectivity for Vκ1 of the immunoglobulin-binding protein.

[0023] "Increased binding selectivity for Vκ1" can refer to a relatively increased binding affinity for Vκ1 relative to the binding affinity for other subgroups of κ light chains. The binding affinity for Vκ1 can be relatively increased relative to the binding affinity for one or more other subgroups of κ light chains. Examples of increased binding selectivity for Vκ1 include increased binding affinity for Vκ1 or decreased binding affinity for other subgroups of κ light chains. Examples of other subgroups of κ light chains include Vκ3 or Vκ4. "Vκ3" refers to κ light chain subgroup 3, in other words, a light chain belonging to subgroup 3. "Vκ4" refers to κ light chain subgroup 4, in other words, a light chain belonging to subgroup 4. According to one embodiment of the present invention, the protein may, for example, be an immunoglobulin-binding protein whose binding affinity for Vκ1 is relatively increased relative to its binding affinity for Vκ3 and / or Vκ4. According to one embodiment of the present invention, specifically, the protein may be, for example, an immunoglobulin-binding protein whose binding selectivity to Vκ1 is increased by reducing its binding to Vκ3 and / or Vκ4.

[0024] Increasing the binding selectivity for Vκ1 can improve the binding selectivity for immunoglobulins containing Vκ1. "Increased binding selectivity for Vκ1-containing immunoglobulins" refers to a relatively greater binding affinity to immunoglobulins containing Vκ1 compared to those containing lower Vκ1 concentrations. For example, by increasing the binding selectivity for Vκ1, the binding affinity to immunoglobulins containing two Vκ1s can be relatively greater than the binding affinity to immunoglobulins containing one Vκ1 or those not containing Vκ1.

[0025] Specifically, the amino acid substitution at the specific position is at least one amino acid substitution selected from Y42H (this expression means that the amino acid residue corresponding to the tyrosine at position 42 of sequence number 1 is replaced with histidine; the same applies to other amino acid substitutions below), K22E, K38D, K38P, Y42F, Y42W, and N44P.

[0026] In other words, the amino acid substitution at the specific position is at least selected from one or more amino acids from (1) to (5): (1) Y42H (2) K22E (3) K38D or K38P (4) Y42F or Y42W (5) N44P. That is, according to one embodiment of the present invention, the protein may have one or more amino acid substitutions selected from (1) to (5). According to one embodiment of the present invention, the protein may, for example, have one, two, three, or four amino acid substitutions selected from (1) to (5). Wherein, amino acid substitutions at the same position in the amino acid substitutions of (1) to (5) are not selected simultaneously. For example, amino acid substitutions of (1) and amino acid substitutions of (4) are not selected simultaneously.

[0027] Of the amino acid substitutions in (1) to (5), the amino acid substitution (Y42H) in (1) is an amino acid substitution that particularly enhances the binding selectivity for immunoglobulins containing the light chain (Vκ1) belonging to subgroup 1. Therefore, immunoglobulin-binding proteins having at least the amino acid substitution (Y42H) in (1) are preferred examples of the proteins of the present invention. That is, according to one embodiment of the present invention, the protein may, for example, have at least the amino acid substitution (Y42H) in (1). Specifically, the protein of one embodiment of the present invention may, for example, have only the amino acid substitution (Y42H) in (1) to (5), or may have a combination of the amino acid substitution (Y42H) in (1) and more than one (e.g., any one, two, or three) of the amino acid substitutions selected from (2), (3), and (5).

[0028] In one embodiment, the amino acid substitution at the specific position may include one or more amino acid substitutions selected from (1) to (5).

[0029] According to one embodiment of the present invention, when a protein has two or more amino acid substitutions, the combination of these amino acid substitutions is not particularly limited.

[0030] As one way to combine amino acid substitutions selected from the amino acid substitutions described in (1) to (5), examples include amino acid substitutions of Y42H and K38D.

[0031] In addition, as another way of combining amino acid substitutions, examples include combinations of amino acid substitutions selected from at least one of (1) to (5) and substitutions with basic amino acids other than lysine (arginine (R) or histidine (H)) or amino acids with hydroxyl groups (serine (S), threonine (T) or tyrosine (Y)) of at least one of the lysine (K) residues selected from (I) to (VII). (I) Lysine residue corresponding to position 7 of sequence number 1 (II) Lysine residue corresponding to position 13 of sequence number 1 (III) Lysine residue corresponding to position 22 of sequence number 1 (IV) Lysine residue corresponding to position 29 of sequence number 1 (V) The lysine residue corresponding to position 38 of sequence number 1 (VI) Lysine residue corresponding to position 48 of sequence number 1 (VII) Lysine residue corresponding to position 67 of sequence number 1

[0032] According to one embodiment of the present invention, the protein may have one, two, three, four, five, six or seven lysine residues substituted.

[0033] Specifically, when the amino acid substitution of (III) is selected, the substitution of the lysine residue of (III) covers the amino acid substitution of (2) K22E; when the amino acid substitution of (V) is selected, the substitution of the lysine residue of (V) covers the amino acid substitution of (3) K38E or K38P.

[0034] Alternatively, the amino acid substitutions in (I) to (VII) may not be selected at the same position as the amino acid substitutions selected from one or more of the amino acid substitutions in (1) to (5). That is, for example, if at least the amino acid substitution of (2) K22E is selected from the amino acid substitutions in (1) to (5), the amino acid substitution in (III) may not be selected.

[0035] Furthermore, the combination of amino acid substitutions is selected such that at least one of the amino acid substitutions from (1) to (5) remains. That is, for example, if the amino acid substitution of (2) K22E is selected alone from the amino acid substitutions from (1) to (5), the amino acid substitution of (III) cannot be selected.

[0036] Furthermore, lysine residues other than those substituted with basic amino acids or amino acids with hydroxyl groups (i.e., residues not selected among the lysine residues in (I) to (VII)) may remain unsubstituted (lysine residues) or may be substituted with amino acids other than basic amino acids and amino acids with hydroxyl groups. Examples of the latter include lysine residues substituted with glutamine or isoleucine, or amino acids of (2) K22E and (3) K38D or K38P mentioned above.

[0037] Furthermore, according to one embodiment of the present invention, the protein may also have at least one other amino acid substitution. Examples of other amino acid substitutions include the following <1> to <53> amino acid substitutions. The following <1> to <53> amino acid substitutions are amino acid substitutions that improve the basic stability of immunoglobulin-binding proteins. According to one embodiment of the present invention, as long as the protein has immunoglobulin-binding activity, for example, it may have one, two, three, four, five, six, seven, eight, or nine or fewer <1> to <53> amino acid substitutions, or it may have more than ten. Wherein, amino acid substitutions at the same position among the following <1> to <53> amino acid substitutions are not selected simultaneously. For example, amino acid substitutions of <1> and amino acid substitution of <18> are not selected simultaneously. <1> N50Y <2> E1G or E1V <3>P3S <4> E4K or E4G <5>E5V <6>K7A <7>E8G <8>E9V <9>I17F <10>G21R <11> E27G or E27R <12> K29P <13>T31L <14>T36A <15>A41T <16> N44S or N44G <17> E49T or E49I <18> Any one of N50S, N50K and N50D <19>G51V <20> Any one of E52V, E52G, and E52D <21>Y53F <22> T54I or T54M <23> Any one of N62H, N62R, N62L, N62M, and N62W <24>N65Y <25>A69T <26> T2S or T2P <27> P3R <28> E5K or E5R <29> Any one of E27V, E27K and E27I <30>F32L <31> K38A <32>N44I <33> A47T or A47R <34>E52N <35>T2A <36>E5D <37> P6L or P6T <38> K7P <39>V14A <40> I23R or I23V <41> K29F <42>T31M <43> Any one of E33D, E33G, and E33V <44> A35T <45>T36S <46> A37T <47> A41I or A41Y <48> N44H or N44R <49> E52L or E52Y <50> G60R <51> I64L <52>I66V <53> A69L or A69V

[0038] According to one embodiment of the invention, the protein may particularly have one or more amino acid substitutions selected from (6) to (11) below. According to one embodiment of the invention, the protein may also particularly have amino acid substitutions of (6) below and amino acid substitutions selected from (7) to (11) below. (6) N50Y (which is an amino acid substitution of the <1>) (7) E4G (which is one of the options for the amino acid substitution of the <4>) (8) K7A (which is a substitution of the amino acid in the <6>) (9) E52L (which is one of the options for amino acid substitution of the <49>) (10) Y53F (which is the amino acid substitution of the <21>) (11) A69V (which is one of the options for amino acid substitution of the <53>)

[0039] Additionally, as another embodiment of the amino acid substitution, the following <54> to <63> amino acid substitutions can be cited. Immunoglobulin-binding proteins having the following <54> to <63> amino acid substitutions can elute immunoglobulins (antibodies) bound to the protein under milder pH conditions (i.e., near-neutral conditions). According to one embodiment of the invention, as long as the protein has immunoglobulin-binding activity, it can, for example, have one, two, three, four, five, six, seven, eight, or nine or fewer <54> to <64> amino acid substitutions, or it can have all 10. <54> K7Q <55> K13V <56> K29V or K29I <57> P6A <58>E8R <59> N15V <60> I23F or I23L <61> Any one of E34D, E34F, E34L, and E34V <62> K38L <63> N50M <64>Y53S

[0040] According to one embodiment of the present invention, the protein may specifically have the following (12) amino acid substitutions. (12) K29I (which is one of the options for amino acid substitution of the <56>)

[0041] Specifically, when the amino acid substitution of <31> or <62> is selected, the amino acid substitution of <31> or <62> covers the amino acid substitution of (3) K38E or K38P; when the amino acid substitution of any one of <16>, <32> and <48> is selected, the amino acid substitution of (5) N44P is covered by the amino acid substitution of <16>, <32> and <48>.

[0042] Alternatively, the amino acid substitutions in <1> to <64> that are at the same position as one or more amino acid substitutions selected from (1) to (5) may not be selected. That is, for example, if at least one amino acid substitution of (3) K38E or K38P is selected from the amino acid substitutions in (1) to (5), the amino acid substitutions in <31> or <62> may not be selected.

[0043] Furthermore, the combination of amino acid substitutions is selected such that at least one or more amino acids selected from the amino acid substitutions of (1) to (5) remain. That is, for example, if only the amino acid substitution of (3) K38E or K38P is selected from the amino acid substitutions of (1) to (5), the amino acid substitution of <31> or <62> cannot be selected.

[0044] In addition, as another type of amino acid substitution, the following amino acid substitutions from to <g> can be cited. According to one embodiment of the present invention, as long as the protein has immunoglobulin binding activity, it may, for example, have one, two, or three or fewer amino acid substitutions from to <d>. Specifically, amino acid substitutions at the same position among the following amino acid substitutions from to <g> are not selected simultaneously. For example, amino acid substitutions of and <c> are not selected simultaneously. E49D P6S <c>E49V <d>N62Y

[0045] Another example of a combination of amino acid substitutions can be cited: Amino acid substitutions of K7A, K13R, K22R, K29I, K38E, Y42H, K48R and K67R; Amino acid substitutions of K7A, K13R, K22E, K29I, K38E, K48R and K67R; Amino acid substitutions of K7A, K13R, K22R, K29I, K38P, K48R and K67R; Amino acid substitutions of K7A, K13R, K22R, K29I, K38D, K48R and K67R; Amino acid substitutions of K7A, K13R, K22R, K29I, K38E, Y42F, K48R and K67R; Amino acid substitutions of K7A, K13R, K22R, K29I, K38E, Y42W, K48R and K67R; Amino acid substitutions of K7A, K13R, K22R, K29I, K38E, N44P, K48R and K67R; Amino acid substitutions for K7A, K13R, K22R, K29I, K38D, Y42H, K48R, and K67R.

[0046] Another example of a combination of amino acid substitutions can be cited: Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42H, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22E, K29I, K38E, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38P, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38D, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42W, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42F, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38E, N44P, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R and A69V; Amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38D, Y42H, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V.

[0047] Also having the other amino acid substitutions described above (i.e., the amino acid substitutions of <1> to <64> and to <d>, as explained below), according to one embodiment of the invention, the protein can be a protein that exhibits improved binding selectivity to antibodies having a specific light chain (e.g., Vκ1) and also improved base stability. An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 100), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42H, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 95), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22E, K29I, K38E, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 96), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38P, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 97), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38D, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 98), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42W, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 99), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38E, Y42F, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 101), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38E, N44P, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V; An immunoglobulin-binding protein (an immunoglobulin-binding protein comprising the amino acid sequence described in sequence number 102), comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence contains substitutions of the amino acids E4G, P6S, K7A, K13R, K22R, K29I, K38D, Y42H, K48R, E49D, N50Y, G52L, Y53F, N62Y, K67R, and A69V.

[0048] In this specification, an amino acid sequence having the amino acid substitutions illustrated above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above) in the amino acid sequence described in Serial No. 1 is also referred to as a "substituted amino acid sequence derived from Serial No. 1". In other words, a substituted amino acid sequence derived from Serial No. 1 is an amino acid sequence described in Serial No. 1 that has undergone the amino acid substitutions illustrated above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above). Furthermore, a substituted amino acid sequence derived from Serial No. 1 is, in other words, an amino acid sequence identical to the amino acid sequence described in Serial No. 1 except for having the amino acid substitutions illustrated above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above). A substituted amino acid sequence derived from Serial No. 1 is an example of a modified amino acid sequence in this specification.

[0049] As a modified amino acid sequence in this specification, variant sequences of the modified amino acid sequence (e.g., derived from the substituted amino acid sequence of Serial Number 1) illustrated above can also be cited. That is, according to one embodiment of the present invention, as a protein, a variant sequence comprising the modified amino acid sequence (e.g., derived from the substituted amino acid sequence of Serial Number 1) illustrated above, and having immunoglobulin binding activity, can also be cited. Hereinafter, the case of a variant sequence derived from the substituted amino acid sequence of Serial Number 1 will be illustrated, and this description can also be applied to variant sequences of any modified amino acid sequence.

[0050] Variant sequences derived from the substituted amino acid sequence of Serial Number 1 are configured such that the amino acid substitution at the specific position remains (i.e., according to one embodiment of the invention, the protein has the amino acid substitution at the specific position). Alternatively, variant sequences derived from the substituted amino acid sequence of Serial Number 1 may also be configured such that other amino acid substitutions described above remain (i.e., according to one embodiment of the invention, the protein has the other amino acid substitutions described above). Furthermore, variant sequences derived from the substituted amino acid sequence of Serial Number 1 may, for example, include one or more amino acid substitutions selected from the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any of the other amino acid substitutions described above) that are not present in the substituted amino acid sequence derived from Serial Number 1. For example, if the substituted amino acid sequence derived from Serial Number 1 does not have the other amino acid substitutions described above, the variant sequence derived from the substituted amino acid sequence of Serial Number 1 may also have the other amino acid substitutions.

[0051] As a variant sequence derived from the substituted amino acid sequence of Serial Number 1, examples include amino acid sequences containing substitutions, deletions, insertions, and / or additions of one or more amino acid residues at one or more positions in the substituted amino acid sequence derived from Serial Number 1. That is, according to one embodiment of the present invention, as long as a protein has immunoglobulin binding activity, it may also contain, in addition to the amino acid substitutions exemplified above (i.e., amino acid substitutions at the specific positions and any other amino acid substitutions described above), substitutions, deletions, insertions, and / or additions of one or more amino acid residues at one or more positions in the amino acid sequence described in Serial Number 1. That is, according to one embodiment of the present invention, as a protein, examples include proteins that contain the amino acid sequence described in Serial Number 1, wherein the amino acid sequence has the amino acid substitutions exemplified above (i.e., amino acid substitutions at the specific positions and any other amino acid substitutions described above), and also contain substitutions, deletions, insertions, and / or additions of one or more amino acid residues at one or more positions, and the protein has immunoglobulin binding activity. In other words, according to one embodiment of the present invention, the protein may, for example, be a protein containing the following amino acid sequence and having immunoglobulin binding activity, wherein the amino acid sequence is identical to Serial No. 1 except for having the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions exemplified above), and also includes substitutions, deletions, insertions, and / or additions of one or more amino acid residues at one or more positions. Furthermore, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues are selected such that the amino acid substitution at the specific position remains. That is, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues may occur, for example, at positions other than the specific position. Additionally, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues may also be selected such that the other amino acid substitutions remain. That is, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues may occur, for example, at positions other than the other amino acid substitutions. Furthermore, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues may, for example, include one or more amino acid substitutions selected from the amino acid substitutions exemplified above, originating from the substituted amino acid sequence of Serial No. 1 that are not present. The term "one or more" varies depending on the position of the amino acid residues in the protein's three-dimensional structure or the type of amino acid residues. Specifically, it can refer to any one of the following: more than 1 and less than 20, more than 1 and less than 10, more than 1 and less than 9, more than 1 and less than 8, more than 1 and less than 7, more than 1 and less than 6, more than 1 and less than 5, more than 1 and less than 4, more than 1 and less than 3, more than 1 and less than 2, or 1.As an example of the substitution of the aforementioned amino acid residues, conserved substitutions can be cited, which occur between amino acids with similar physical and / or chemical properties. In the case of conserved substitutions, those skilled in the art know that protein function is generally maintained between the substituted and unsubstituted proteins. Examples of conserved substitutions include substitution between glycine and alanine, between serine and threonine, or between glutamic acid and aspartic acid (Structure and Function of Proteins, MEDICAL SCIENCES INTERNATIONAL, 9, 2005). Furthermore, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues also include substitutions, deletions, insertions, and / or additions resulting from naturally occurring variations (mutations or variants) such as individual differences or species differences based on the microorganism from which the protein or the gene encoding it originates.

[0052] Furthermore, as variant sequences derived from the substituted amino acid sequence of Serial No. 1, examples of amino acid sequences with high homology to the substituted amino acid sequence of Serial No. 1 can also be cited. That is, according to one embodiment of the present invention, as a protein, examples of proteins can also be cited that contain amino acid sequences with high homology to the amino acid sequences having the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above) in the amino acid sequence described in Serial No. 1, and have immunoglobulin binding activity. In addition, the changes in the amino acid sequence within the homology range described above are selected in a manner that leaves behind the amino acid substitution at the specific position. That is, the changes in the amino acid sequence within the homology range described above can, for example, occur at positions other than the specific position. In addition, the changes in the amino acid sequence within the homology range described above can also, for example, include one or more amino acid substitutions selected from the amino acid substitutions exemplified above that are not present in the substituted amino acid sequence derived from Serial No. 1. The term "homology" can refer to similarity or identity, particularly identity. "Homology relative to the amino acid sequence" refers to homology relative to the overall amino acid sequence. "High homology" can refer to homology of 70%, 80%, 90%, or 95%. "Identity" between amino acid sequences refers to the ratio of amino acid residues of the same type in these sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). "Similarity" between amino acid sequences refers to the sum of the ratio of amino acid residues of the same type and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). Regarding amino acid residues with similar side chain properties, the above applies. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0053] Furthermore, as a modified amino acid sequence, examples can be given of amino acid sequences that, in addition to the variant sequences exemplified above, also have one or more amino acid substitutions selected from the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above). For example, a modified amino acid sequence may be an amino acid sequence that, in addition to the variant sequence having at least the amino acid substitution at the specific position, also has other amino acid substitutions.

[0054] Furthermore, as unmodified amino acid sequences, variant sequences of the unmodified amino acid sequences illustrated above (e.g., the amino acid sequence described in Serial No. 1) can also be cited. That is, as modified amino acid sequences, amino acid sequences having the amino acid substitutions illustrated above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above) in variant sequences of the unmodified amino acid sequences illustrated above (e.g., the amino acid sequence described in Serial No. 1) can also be cited as proteins: proteins comprising variant sequences of the unmodified amino acid sequences illustrated above (e.g., the amino acid sequence described in Serial No. 1), wherein the variant sequences have the amino acid substitutions illustrated above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above), and the protein has immunoglobulin binding activity. In other words, according to an embodiment of the present invention, a protein may also be, for example, a protein comprising an amino acid sequence identical to the variant sequence of the unmodified amino acid sequences illustrated above (e.g., the amino acid sequence described in Serial No. 1), except for having the amino acid substitutions illustrated above. Furthermore, variant sequences of the unmodified amino acid sequences exemplified above (such as the amino acid sequence described in Serial No. 1) may or may not actually exist in *Fingoldii* bacteria. That is, "the amino acid sequence of the immunoglobulin-binding domain of protein L derived from *Fingoldii* bacteria (the amino acid sequence of the immunoglobulin-binding domain of FpL)" is not limited to the amino acid sequence of the immunoglobulin-binding domain of protein L actually existing in *Fingoldii* bacteria, but also includes (i.e., hypothetical) amino acid sequences that are variant sequences of this amino acid sequence but do not actually exist in *Fingoldii* bacteria. Hereinafter, examples of variant sequences of the amino acid sequence described in Serial No. 1 will be given, and this explanation can also be applied to variant sequences of any unmodified amino acid sequence.

[0055] As a variant sequence of the amino acid sequence described in Serial No. 1, examples include amino acid sequences in which one or more amino acid residues at one or more positions are substituted, deleted, inserted, and / or added. That is, according to one embodiment of the invention, as a protein, examples include proteins comprising the amino acid sequence described in Serial No. 1, wherein the amino acid sequence contains substitutions, deletions, insertions, and / or additions of one or more amino acid residues at one or more positions, and also has the amino acid substitutions exemplified above (i.e., amino acid substitutions at the specific positions and any other amino acid substitutions described above), and the protein has immunoglobulin binding activity. The substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues can occur, for example, at positions other than the specific positions. Furthermore, the substitutions, deletions, insertions, and / or additions of the aforementioned amino acid residues can also occur, for example, at positions other than other amino acid substitutions.

[0056] As variant sequences of the amino acid sequence described in Serial No. 1, examples of amino acid sequences with high homology to the amino acid sequence described in Serial No. 1 can also be cited. That is, according to one embodiment of the present invention, as a protein, examples of proteins can also be cited that comprise an amino acid sequence with high homology to the amino acid sequence described in Serial No. 1, wherein the amino acid sequence (the amino acid sequence with high homology to the amino acid sequence described in Serial No. 1) has the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions described above), and the protein has immunoglobulin binding activity. Changes in amino acid residues within the homology range described above can, for example, occur at positions other than the specific position. Furthermore, changes in amino acid residues within the homology range described above can, for example, occur at positions other than other amino acid substitutions.

[0057] Other variant sequences of the amino acid sequence described in Serial No. 1 may follow the description of variant sequences of substituted amino acid sequences derived from Serial No. 1.

[0058] In this specification, "the amino acid at position X in the amino acid sequence described in Serial No. 1" refers to the amino acid located at position X, counting from the N-terminus of the amino acid sequence described in Serial No. 1. "The amino acid residue corresponding to the amino acid at position X in the amino acid sequence described in Serial No. 1" in a specific amino acid sequence refers to an amino acid residue in that specific amino acid sequence that, in the alignment of that specific amino acid sequence with the amino acid sequence of Serial No. 1, is positioned at the same position as the amino acid at position X in the amino acid sequence shown in Serial No. 1. For example, in the case of amino acid substitution at Y42H, "the amino acid residue corresponding to the tyrosine at position 42 in Serial No. 1" in a specific amino acid sequence refers to an amino acid residue in that specific amino acid sequence that, in the alignment of that specific amino acid sequence with the amino acid sequence of Serial No. 1, is positioned at the same position as the tyrosine at position 42 in the amino acid sequence shown in Serial No. 1. Furthermore, the "amino acid residue corresponding to the amino acid at position X in the amino acid sequence described in Serial No. 1" in the amino acid sequence described in Serial No. 1 refers to the amino acid at position X itself in the amino acid sequence described in Serial No. 1. That is, the position of the amino acid substitutions exemplified above (i.e., the amino acid substitution at the specific position and any other amino acid substitutions) does not necessarily represent the absolute position in the protein of one embodiment of the present invention, but rather the relative position based on the amino acid sequence described in Serial No. 1. That is, for example, if the protein of one embodiment of the present invention contains an insertion, deletion, or addition of an amino acid residue on the N-terminal side of the position of the amino acid substitution exemplified above, the absolute position of the amino acid substitution may change accordingly. According to one embodiment of the present invention, the position of the amino acid substitution exemplified above in the protein can be determined, for example, by comparing the amino acid sequence of the protein of one embodiment of the present invention with the amino acid sequence described in Serial No. 1. The comparison can be performed, for example, by an alignment program such as BLAST or FASTA. The same applies to the position of the amino acid substitution exemplified above in any amino acid sequence, such as a variant sequence of the amino acid sequence described in Serial No. 1. In addition, the amino acid residues preceding the substitution in the above-described amino acid substitutions (i.e., the amino acid substitutions at the specific positions and any other amino acid substitutions described above) represent the types of amino acid residues preceding the substitution in the amino acid sequence recorded in Serial No. 1. These residues may be retained in unmodified amino acid sequences other than those recorded in Serial No. 1, or they may not be retained.

[0059] According to one embodiment of the present invention, a protein may contain only one modified amino acid sequence or may contain multiple modified amino acid sequences. According to one embodiment of the present invention, a protein may contain, for example, two or more, three or more, four or more, or five or more modified amino acid sequences, or fewer than 10, seven or fewer, five or fewer, four or fewer, three or fewer, or two or fewer modified amino acid sequences, and may also contain a number of non-contradictory combinations thereof. According to one embodiment of the present invention, when a protein contains multiple modified amino acid sequences, the amino acid sequences of these multiple modified amino acid sequences may be the same or different. These multiple modified amino acid sequences may be directly linked (directly connected) or linked via a suitable linker (e.g., an oligopeptide composed of 5 or more and 25 or fewer amino acid residues).

[0060] According to one embodiment of the present invention, a protein may consist of a modified amino acid sequence and may also contain other amino acid sequences. That is, according to one embodiment of the present invention, a protein may, for example, also contain other amino acid sequences at its N-terminus or C-terminus. In other words, according to one embodiment of the present invention, other amino acid sequences may be added to the protein, for example, at the N-terminus or C-terminus of the modified amino acid sequence. Examples of other amino acid sequences include oligopeptides. Examples of oligopeptides that are not linked to the linker are also possible. According to one embodiment of the present invention, there are no particular limitations on the other amino acid sequences as long as they do not impair the immunoglobulin-binding activity or stability of the protein. For example, according to one embodiment of the present invention, there are no particular limitations on the type or length of the other amino acid sequences as long as they do not impair the immunoglobulin-binding properties or stability of the protein.

[0061] According to one embodiment of the present invention, the protein may, for example, contain portions of other immunoglobulin-binding domains in addition to the selected immunoglobulin-binding domain. For example, according to one embodiment of the present invention, when the protein contains a modified amino acid sequence of the FpL domain C3, the protein of one embodiment of the present invention may further contain a portion of the N-terminal side region (domain C1, domain C2) of the FpL domain C3, and may also further contain a portion of the C-terminal side region (domain C4) of the FpL domain C3.

[0062] According to one embodiment of the invention, the protein may also contain, for example, oligopeptides useful for the specific detection or separation of a target analyte on its N-terminal or C-terminal side. Examples of such oligopeptides include polyhistidine and polyarginine. Furthermore, according to one embodiment of the invention, the protein may also contain, for example, oligopeptides useful for immobilizing the protein on a solid phase such as a chromatographic support on its N-terminal or C-terminal side according to an embodiment of the invention. Examples of such oligopeptides include oligopeptides containing lysine and cysteine.

[0063] According to one embodiment of the present invention, when a protein contains other amino acid sequences as described above, the protein of one embodiment of the present invention can be manufactured, for example, by pre-containing other amino acid sequences, or by adding separately manufactured other amino acid sequences as described above. According to one embodiment of the present invention, when a protein contains other amino acid sequences as described above, typically, the protein of one embodiment of the present invention can be manufactured by expressing a polynucleotide encoding the full-length amino acid sequence of the protein of one embodiment of the present invention containing the other amino acid sequences as described above. That is, for example, a polynucleotide encoding other amino acid sequences and a polynucleotide encoding a protein of one embodiment of the present invention (e.g., a protein not containing other amino acid sequences) can be linked by adding other amino acid sequences to the N-terminal or C-terminal side of the protein of one embodiment of the present invention, thereby expressing the protein of one embodiment of the present invention. Additionally, for example, chemically synthesized other amino acid sequences can be chemically bonded to the N-terminal or C-terminal side of the protein of one embodiment of the present invention (e.g., a protein not containing other amino acid sequences).

[0064] According to one embodiment of the present invention, a protein can be manufactured, for example, by expression of a polynucleotide encoding a protein of an embodiment of the present invention. In this specification, the polynucleotide encoding a protein of an embodiment of the present invention will also be referred to as "a polynucleotide of an embodiment of the present invention". Specifically, a polynucleotide of an embodiment of the present invention may be a polynucleotide comprising a nucleotide sequence encoding a protein of an embodiment of the present invention.

[0065] The polynucleotide of one embodiment of the present invention can be obtained, for example, by chemical synthesis, DNA amplification methods such as PCR. The DNA amplification method can be carried out using a polynucleotide containing a nucleotide sequence to be amplified, such as a nucleotide sequence encoding a protein, as a template. Examples of polynucleotides that can be used as templates include genomic DNA of an organism expressing a protein of one embodiment of the present invention, cDNA of a protein of one embodiment of the present invention, and a vector containing the polynucleotide of one embodiment of the present invention.

[0066] The nucleotide sequence of the polynucleotide of one embodiment of the present invention can be designed, for example, by converting the amino acid sequence of the protein of one embodiment of the present invention. When converting from an amino acid sequence to a nucleotide sequence, a standard codon table can be used, preferably considering the frequency of codon usage in the host in which the polynucleotide of one embodiment of the present invention is converted. For example, in the case of Escherichia coli, the frequency of use of AGA / AGG / CGG / CGA for arginine (R), ATA for isoleucine (I), CTA for leucine (L), GGA for glycine (G), and CCC for proline (P) (so-called rare codons) can be avoided during the conversion. Analysis of codon usage frequencies can also be performed using public databases (e.g., the Codon Usage Database on the website of the Kazusa DNA Institute).

[0067] The polynucleotide of one embodiment of the present invention can be obtained in its full length in one go, or it can be obtained by ligating a polynucleotide composed of a partial sequence of the polynucleotide. The description of the method for obtaining the polynucleotide of one embodiment of the present invention as described above is not limited to the case of obtaining its full-length sequence in one go, but can also be applied to the case of obtaining its partial sequence.

[0068] Furthermore, the polynucleotides in one embodiment of the present invention can also be designed by linking to a polynucleotide encoding an amino acid sequence of any polypeptide, thereby encoding a fusion-type amino acid sequence.

[0069] The protein of one embodiment of the present invention can be manufactured, for example, by culturing a gene recombinant host containing the polynucleotide of one embodiment of the present invention (hereinafter also simply referred to as "the gene recombinant host of one embodiment of the present invention") and causing it to express the protein, and then recovering the expressed protein. The gene recombinant host of one embodiment of the present invention can be obtained, for example, by using a polynucleotide transformation host of one embodiment of the present invention. There are no particular limitations as long as the host can express the protein of one embodiment of the present invention by transformation with the polynucleotide of one embodiment of the present invention. Examples of hosts include animal cells, insect cells, and microorganisms. Examples of animal cells include COS cells, CHO (Chinese Hamster Ovary) cells, HeLa cells, NIH3T3 cells, and HEK293 cells; examples of insect cells include Sf9 cells and BTI-TN-5B1-4 cells; and examples of microorganisms include yeast and bacteria. Furthermore, examples of yeasts include *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Schizosaccharomyces pombe*. Examples of bacteria include *Escherichia coli*. Examples of *Escherichia coli* strains include JM109 and BL21(DE3). Moreover, using yeast or *E. coli* as a host is preferred in terms of productivity, with *E. coli* being even more preferred.

[0070] In a gene recombination host according to one embodiment of the present invention, the polynucleotide of the present invention can be maintained in an expressible manner. Specifically, the polynucleotide of the present invention can be maintained in a manner controlled by a promoter that functions in the host. In the case that the host is *Escherichia coli*, examples of promoters that function in the host include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.

[0071] Furthermore, in the gene recombination host of one embodiment of the present invention, the polynucleotide of one embodiment of the present invention can exist on a vector that autonomously replicates outside the genomic DNA. That is, the gene recombination host of one embodiment of the present invention can also be created by transforming a host with an expression vector containing the polynucleotide of one embodiment of the present invention (hereinafter also simply referred to as "the expression vector of one embodiment of the present invention"). The expression vector of one embodiment of the present invention is obtained, for example, by inserting the polynucleotide of one embodiment of the present invention at an appropriate position in the expression vector. In addition, there are no particular limitations as long as the expression vector can exist and replicate stably in the transformed host. In the case of Escherichia coli, pET plasmid vector, pUC plasmid vector, and pTrc plasmid vector can be cited as examples of expression vectors. In addition, the expression vector of one embodiment of the present invention can have selection markers such as antibiotic resistance genes. In addition, the appropriate position refers to the position that does not destroy the replication function, selection markers, or regions related to transmissibility of the expression vector. When inserting the polynucleotide of one embodiment of the present invention into the expression vector, it is preferable to insert it in a state linked to a functional polynucleotide such as a promoter required for expression.

[0072] Furthermore, in the gene recombination host of one embodiment of the present invention, the polynucleotides of one embodiment of the present invention can also be introduced into genomic DNA. The introduction of polynucleotides into genomic DNA according to one embodiment of the present invention can be carried out, for example, using a gene introduction method based on homologous recombination. Examples of gene introduction methods based on homologous recombination include red-driven integration (Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA., 2000; 97: 6640-6645) using linear DNA, methods using vectors containing temperature-sensitive origins of replication, methods using vectors without origins of replication that function within the host, and phage transduction.

[0073] Transformation of a host containing a polynucleotide such as an expression vector according to an embodiment of the present invention can be carried out, for example, by methods commonly used by those skilled in the art. For example, when *E. coli* is selected as the host, transformation can be performed using competent cell methods, heat shock methods, electroporation methods, etc. After transformation, by screening for hosts containing the polynucleotides of an embodiment of the present invention using appropriate methods, a gene recombination host according to an embodiment of the present invention can be obtained.

[0074] In addition, information related to genetic engineering methods, such as expression vectors and promoters that can be used in various microorganisms, can be obtained from well-known literature such as "Lectures on Microbiology 8: Genetic Engineering, Kyoritsu Publishing, 1987".

[0075] In the case where the gene recombination host of one embodiment of the present invention contains the expression vector of one embodiment of the present invention, the expression vector of one embodiment of the present invention can be prepared from the gene recombination host of one embodiment of the present invention. For example, the expression vector of one embodiment of the present invention can be prepared from the culture obtained by culturing the gene recombination host of one embodiment of the present invention using alkaline extraction or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0076] By culturing a recombinant host according to an embodiment of the present invention, the protein according to an embodiment of the present invention can be expressed. Furthermore, by culturing the aforementioned recombinant host to express the aforementioned protein and recovering the expressed protein, the aforementioned protein can be manufactured. That is, this specification discloses a method for manufacturing the aforementioned protein, comprising: a step of culturing the aforementioned recombinant host to express the aforementioned protein; and a step of recovering the expressed protein. The composition of the culture medium and the culture conditions can be appropriately set according to various conditions such as the type of host and the characteristics of the aforementioned protein. For example, the composition of the culture medium and the culture conditions can be set to conditions that allow the host to proliferate and express the aforementioned protein. For example, a culture medium appropriately containing a carbon source, a nitrogen source, inorganic salts, and various other organic and inorganic components can be used. Specifically, when the host is *Escherichia coli*, LB (Luria-Bertani) medium (1% tryptone (w / v), 0.5% yeast extract (w / v), 1% NaCl) supplemented with necessary nutrients is a preferred example of a culture medium. Furthermore, in order to enable the recombinant host of one embodiment of the present invention to selectively proliferate depending on whether the expression vector of one embodiment of the present invention has been introduced, it is preferable to add an antibiotic corresponding to the antibiotic resistance gene contained in the expression vector to the culture medium and then culture it. For example, if the expression vector contains a kanamycin resistance gene, kanamycin can be added to the culture medium. The same applies when a polynucleotide of one embodiment of the present invention is introduced into genomic DNA. In addition, the culture medium may also contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystatin, thioglycolate, and dithiothreitol. In addition, the culture medium may also contain a reagent such as glycine that promotes the secretion of proteins from the transformant into the culture medium. For example, when the host is *Escherichia coli*, it is preferable to add 2% (w / v) or less of glycine relative to the culture medium. For example, when the host is *Escherichia coli*, the culture temperature is generally 10°C or higher and 40°C or lower, preferably 20°C or higher and 37°C or lower, and more preferably around 25°C. For example, when the host is *Escherichia coli*, the pH of the culture medium is between pH 6.8 and pH 7.4, preferably around pH 7.0. Furthermore, when expressing the above protein under the control of an inducible promoter, induction is preferred to ensure good expression of the protein. In expression induction, for example, an inducer corresponding to the type of promoter can be used. An example of an inducer is IPTG (Isopropyl-β-D-thiogalactopyranoside).When the host is *Escherichia coli*, for example, the expression of the aforementioned protein can be induced by adding an appropriate amount of IPTG when the turbidity (absorbance at 600 nm) of the culture medium reaches approximately 0.5 to 1.0, and continuing culturing. The concentration of IPTG added is, for example, 0.005 mM or more and 1.0 mM or less, preferably 0.01 mM or more and 0.5 mM or less. Expression induction, such as IPTG induction, can be performed under conditions known in the art.

[0077] The protein of one embodiment of the present invention can be isolated and recovered from the culture by a method suitable for its expression mode. Furthermore, in this specification, "culture" refers to the whole or a portion of the culture medium obtained by culturing. This portion is not particularly limited as long as it contains the aforementioned protein. Examples of this portion include cells of the transformed organism of one embodiment of the present invention cultured, and the culture medium (i.e., culture supernatant). For example, if the aforementioned protein accumulates in the culture supernatant, the cells can be separated by centrifugation, and the protein can be recovered from the obtained culture supernatant. Alternatively, if the aforementioned protein accumulates within cells (including the periplasm), after recovering the cells by centrifugation, an enzyme treatment agent, surfactant, etc., is added to lyse the cells, and the protein can be recovered from the lysate. The above-described recovery of the protein from the culture supernatant and cell lysate can be carried out, for example, by methods known for protein separation and purification. Examples of such recovery methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation.

[0078] The protein of one embodiment of the present invention can be used, for example, for the separation or analysis of immunoglobulins (antibodies). When used for this purpose, for example, it can be used as an immunoglobulin adsorbent comprising an insoluble carrier and the aforementioned protein immobilized on the insoluble carrier (hereinafter also simply referred to as "the immunoglobulin adsorbent of one embodiment of the present invention"). Furthermore, in this specification, "immunoglobulin separation" is not limited to the separation of immunoglobulins from a solution containing impurities, but also includes the separation between immunoglobulins based on structure, properties, or activity. There are no particular limitations on the insoluble carrier, as long as it is insoluble relative to aqueous solutions. Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate, carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides or synthetic polymers are preferred as insoluble carriers. Examples of preferred carriers include polymethyl methacrylate gels such as TOYOPEARL (manufactured by Tosoh Corporation) with introduced hydroxyl groups, agarose gels such as Sepharose (manufactured by Cytiva Corporation), and cellulose gels such as Cellufine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited. For example, the insoluble carrier can be shaped to fill a column. For example, the insoluble carrier can be granular or non-granular. Furthermore, the insoluble carrier can also be porous or non-porous.

[0079] The immunoglobulin (antibody) to be isolated or analyzed in one embodiment of the present invention is not particularly limited as long as it is an immunoglobulin that has binding affinity to the protein of one embodiment of the present invention. Specifically, the immunoglobulin may be an immunoglobulin having a κ light chain. More specifically, the immunoglobulin may be an immunoglobulin having Vκ1. The immunoglobulin may contain other subgroups of κ light chains besides Vκ1. Examples of other subgroups of κ light chains include Vκ3 and Vκ4. The immunoglobulin may contain other regions such as the Fc region besides the κ light chain, or it may not contain them. The immunoglobulin may also be, for example, a single-chain Fv (scFv), Fab, F(ab')2, IgA, bispecific T cell-induced (BiTE) antibody, or other immunoglobulins that do not have an Fc region. The immunoglobulin may be a monoclonal antibody or a polyclonal antibody. The immunoglobulin may also be, for example, any one of IgG, IgM, IgA, IgD, and IgE. IgG may also be, for example, any one of IgG1, IgG2, IgG3, and IgG4. There are no particular restrictions on the source of immunoglobulins. Immunoglobulins can originate from a single organism or from a combination of two or more organisms. Examples of immunoglobulins include chimeric antibodies, humanized antibodies, human antibodies, and their variants (e.g., amino acid substitutes). Additionally, examples of immunoglobulins include bispecific antibodies, fusion antibodies of the κ light chain and other proteins, and complexes of the κ light chain and drugs (ADCs), which are artificially modified antibodies. Furthermore, the term "immunoglobulin" also includes antibody drugs. Bispecific antibodies are particularly noteworthy as immunoglobulins. Examples of bispecific antibodies include trifunctional antibodies, F(ab')2, single-chain Fv (scFv), and bispecific T cell-induced (BiTE) antibodies. Bispecific antibodies can, for example, have Vκ1 and Vκ3, or Vκ1 and Vκ4. Bispecific antibodies can particularly have Vκ1 and Vκ3. By utilizing the protein of one embodiment of the present invention, for example, it is possible to separate immunoglobulins containing Vκ1 into immunoglobulins with higher or lower Vκ1 content compared to their counterparts. Specifically, by utilizing the aforementioned protein, for example, it is possible to separate immunoglobulins containing two Vκ1s into immunoglobulins containing one Vκ1 and immunoglobulins not containing Vκ1. Specifically, by utilizing the aforementioned protein, for example, it is possible to separate immunoglobulins containing one Vκ1 (e.g., bispecific antibodies having Vκ1 and other subgroups of the κ light chain) into immunoglobulins containing two Vκ1s or immunoglobulins not containing Vκ1.

[0080] In one embodiment of the present invention, the protein can also be covalently fixed to an insoluble carrier. Specifically, the protein and the insoluble carrier can be covalently bonded via active groups present in the insoluble carrier, thereby fixing the protein to the insoluble carrier. That is, the insoluble carrier may have active groups on its surface. Examples of such active groups include N-hydroxysuccinimide (NHS) activated ester groups, epoxy groups, carboxyl groups, maleimide groups, haloacetyl groups, trifluoroethanesulfonyl (Tresyl), formyl groups, and haloacetamides. As an insoluble carrier with active groups, a commercially available insoluble carrier with active groups can be used as is, or active groups can be introduced into the insoluble carrier for use. Examples of commercially available carriers with active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HPColumns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (both manufactured by Cytiva Corporation), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0081] As a method for introducing active groups onto the surface of a support, an example is a method in which one site of a compound having two or more active sites reacts with hydroxyl, epoxy, carboxyl, amino, or other groups present on the surface of the support.

[0082] Examples of compounds that introduce epoxy groups into hydroxyl and amino groups present on the surface of a support include epichlorohydrin, ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether, and hexanediol diglycidyl ether.

[0083] In addition, examples of compounds that introduce carboxyl groups into epoxy groups present on the surface of a support include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0084] In addition, examples of compounds that introduce maleimide groups into hydroxyl, epoxy, carboxyl, or amino groups present on the surface of a support include: N-(ε-maleimide hexanoic acid) hydrazide, N-(ε-maleimide propionic acid) hydrazide, 4-(4-N-maleimide phenyl)acetylhydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimide)phenyl isocyanate, and 2-maleimide. Acetic acid, 3-maleimide propionic acid, 4-maleimide butyric acid, 6-maleimide hexanoic acid, N-(α-maleimide acetoxy)succinimide ester, (m-maleimide benzoyl)N-hydroxysuccinimide ester, succinimide-4-(maleimide methyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimide-4-(maleimide methyl)cyclohexane-1-carboxylic acid, (p-maleimide benzoyl)N-hydroxysuccinimide ester, (m-maleimide benzoyl)N-hydroxysuccinimide ester.

[0085] In addition, examples of compounds that introduce halogenated acetyl groups into hydroxyl and amino groups present on the surface of a support include: chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetyl chloride, bromoacetyl chloride, bromoacetyl bromide, chloroacetic anhydride, bromoacetic anhydride, iodoacetic anhydride, 2-(iodoacetamide)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

[0086] Another method for introducing active groups onto the surface of a support can be illustrated by reacting an ω-alkenyl alkyl glycidyl ether with hydroxyl or amino groups present on the surface of the support, followed by halogenating the ω-alkenyl sites with a halogenating agent. Examples of ω-alkenyl alkyl glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0087] In addition, as a method for introducing active groups onto the surface of a support, another example is the method of introducing active groups onto carboxyl groups present on the surface of the support by using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide, and carbonyl diimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

[0088] The immobilization of a protein onto an insoluble carrier according to one embodiment of the present invention can be carried out, for example, in a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set according to various conditions such as the reactivity of the active group and the stability of the protein according to one embodiment of the present invention. The reaction temperature during immobilization can be, for example, 5°C or higher and 50°C or lower, preferably 10°C or higher and 35°C or lower.

[0089] An immunoglobulin adsorbent according to one embodiment of the present invention can be used for the separation of immunoglobulins (antibodies) by forming a column filled with the adsorbent. For example, by adding a solution containing immunoglobulins to a column filled with the immunoglobulin adsorbent, the immunoglobulins are adsorbed onto the adsorbent, and the adsorbed immunoglobulins are eluted, thereby separating the immunoglobulins. That is, this specification discloses a method for separating immunoglobulins contained in the solution, comprising: adding a solution containing immunoglobulins to a column filled with the immunoglobulin adsorbent to adsorb the immunoglobulins onto the adsorbent; and eluting the immunoglobulins adsorbed onto the adsorbent. The solution containing immunoglobulins can be added to the column, for example, using a pump or similar delivery unit. Furthermore, the solution containing immunoglobulins can be pre-solvent-substituted with a suitable buffer solution before being added to the column. Additionally, the column can be equilibrated with a suitable buffer solution before adding the solution containing immunoglobulins to the column. By equilibrating the column, for example, it is expected that immunoglobulins can be separated with higher purity. Examples of buffers used in solvent substitution and equilibration include phosphate buffer, acetate buffer, and MES buffer. In such buffers, inorganic salts such as sodium chloride at a concentration of 1 mM or higher and 1000 mM or lower may be added, for example. The buffer used in solvent substitution and the buffer used in equilibration may be the same or different. Furthermore, if impurities or other components besides immunoglobulins remain in the column after passing the solution containing immunoglobulins through the column, these components can be removed from the column before eluting the immunoglobulins adsorbed on the immunoglobulin adsorbent. Components other than immunoglobulins can be removed from the column, for example, using a suitable buffer. Regarding the buffer used for removing components other than immunoglobulins, for example, the descriptions of buffers used in solvent substitution or equilibration can be followed. Immunoglobulins adsorbed on the immunoglobulin adsorbent can be eluted, for example, by weakening the interaction between the immunoglobulin and the ligand (the protein of one embodiment of the present invention). Methods for weakening the interaction between the immunoglobulin and the ligand (the protein of one embodiment of the present invention) include pH alteration, addition of an antipeptide, temperature increase, and salt concentration change. As a method to reduce the interaction between immunoglobulins and ligands (proteins in one embodiment of the present invention), pH alteration can be particularly exemplified. That is, as a step in eluting immunoglobulins adsorbed on the adsorbent, a step in eluting immunoglobulins adsorbed on the adsorbent by changing the pH can be particularly exemplified. As a pH alteration, a decrease in pH can be exemplified. As a decrease in pH, a decrease in pH achieved by using a buffer solution can be exemplified. Specifically, immunoglobulins adsorbed on the immunoglobulin adsorbent can be eluted, for example, using a suitable eluent.As an eluent, a buffer that is more acidic than the buffer used in solvent substitution and equilibration can be cited. Examples of such acidic buffers include citrate buffer, glycine hydrochloride buffer, and acetate buffer. That is, by using such an acidic buffer, the pH can be lowered. The pH of the eluent can be set, for example, within a range that does not impair the function of immunoglobulins (such as their binding to antigens). The pH of the eluent can be, for example, 2 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3 or higher, 3.1 or higher, or 3.2 or higher, or it can be 4 or lower, 3.5 or lower, 3.2 or lower, 3.1 or lower, 3 or lower, 2.9 or lower, 2.8 or lower, 2.7 or lower, 2.6 or lower, or 2.5 or lower, or combinations thereof. Specifically, the pH of the eluent can be, for example, above 2 and below 4, above 2.2 and below 4, or above 2.4 and below 4.

[0090] Immunoglobulins (antibodies) are separated using the method described above to obtain isolated immunoglobulins. Specifically, in one of these methods, the immunoglobulin separation method can be a method for manufacturing immunoglobulins, and more specifically, a method for manufacturing isolated immunoglobulins. Immunoglobulins can be obtained, for example, as an elution fraction containing immunoglobulins. That is, the fraction containing the eluted immunoglobulins can be separated. The separation of the fraction can be performed, for example, by conventional methods. Examples of methods for separating the fraction include: changing the recovery container at regular intervals or at regular volumes; changing the recovery container according to the shape of the chromatogram of the eluent; and separating the fraction using an automated fraction collector such as an automated sampler. Furthermore, immunoglobulins can also be recovered from the fraction containing immunoglobulins. The recovery of immunoglobulins from the fraction containing immunoglobulins can be performed, for example, using known methods used in protein separation and purification. Example

[0091] Hereinafter, several embodiments of the present invention will be further described in detail using examples and comparative examples, but the present invention is not limited to these examples. Furthermore, the reference examples do not illustrate embodiments of the present invention.

[0092] Example 1: Preparation of the expression vector for the protein L immunoglobulin binding domain C3 (1) A polynucleotide (Sequence No. 2) was synthesized, encoding the amino acid sequence of the immunoglobulin-binding domain C3 of protein L (hereinafter referred to as FpL) of Finegoldia magna, consisting of the amino acid sequence described in Sequence No. 1 (amino acid residues 394 to 463 of GenBank No. AAA67503 (Sequence No. 106), also referred to as "FpL_C3"). In addition, during synthesis, a recognition sequence for the restriction enzyme NcoI (5'-CCATGG-3') was added to the 5' end, and a nucleotide sequence encoding 6 histidine residues (5'-CATCACCACCATCACCAC-3'; Sequence No. 107), a stop codon, and a recognition sequence for the restriction enzyme HindIII (5'-AAGCTT-3') was added to the 3' end.

[0093] (2) The polynucleotide containing sequence number 2 obtained through synthesis was treated with restriction enzymes NcoI and HindIII, and the size of the target band was confirmed by agarose gel electrophoresis. After the target band was excised, the polynucleotide was purified using the QIAquick Gel Extraction kit (QIAGEN).

[0094] (3) The polynucleotides obtained in (2) and the plasmid pET-26b, which had been pre-digested with restriction enzymes NcoI and HindIII, were ligated using a DNA Ligation Kit (manufactured by Takara Bio Inc.). The ligation product was used to transform Escherichia coli BL21 (DE3) strain and cultured on LB (Luria-Bertani) plates containing 50 μg / mL kanamycin (37°C, 16 h).

[0095] (4) Select the transformant (recombinant Escherichia coli) obtained in (3), culture it in LB medium containing 50 μg / mL kanamycin, and then purify it using QIAprepSpin Miniprep kit (QIAGEN) to obtain a vector that can express FpL_C3 (named pET-FpL_C3).

[0096] (5) For the polynucleotide encoding FpL_C3 and its surrounding region in the expression vector pET-FpL_C3 obtained in (4), a Big Dye Terminator Cycle Sequencing ready Reaction kit (Thermo Fisher Scientific) based on chain termination was used for cyclic sequencing reactions, and the nucleotide sequence was analyzed using an ABI Prism 3700 DNA analyzer (Thermo Fisher Scientific). In addition, oligonucleotides consisting of sequences recorded in sequence number 3 (5'-TAATACGACTCACTATAGGG-3') or sequence number 4 (5'-TATGCTAGTTATTGCTCAG-3') were used as sequencing primers for this analysis.

[0097] The sequence analysis confirmed that a polynucleotide consisting of the sequence recorded in sequence number 2 was inserted into the expression vector pET-FpL_C3.

[0098] Example 2: Importing variants into FpL_C3 and creating a library (Part 1) The variant was randomly introduced into the polynucleotide portion (sequence number 2) encoding the immunoglobulin-binding protein in the expression vector pET-FpL_C3 prepared in Example 1 using error-prone PCR.

[0099] (1) Error-prone PCR was performed using pET-FpL_C3 prepared in Example 1 as template DNA. Error-prone PCR was performed as follows: after preparing the reaction solution with the composition shown in Table 1, the reaction was performed for 30 cycles. The reaction solution was then heat-treated at 95°C for 2 minutes. One cycle consisted of a first step of heat treatment at 95°C for 30 seconds, a second step of heat treatment at 50°C for 30 seconds, and a third step of heat treatment at 72°C for 90 seconds. Finally, the reaction solution was heat-treated at 72°C for 7 minutes. The error-prone PCR successfully introduced the mutation into the polynucleotide encoding FpL_C3 (Sequence No. 2), with an average mutation introduction rate of 1.6%.

[0100] [Table 1]

[0101] (2) After purifying the obtained PCR product, it was digested with restriction enzymes NcoI and HindIII and ligated into the expression vector pET-26b which had been digested with restriction enzymes NcoI and HindIII.

[0102] (3) After the ligation reaction was completed, the Escherichia coli BL21 (DE3) strain was transformed with the reaction solution and cultured on LB agar containing 50 μg / mL kanamycin (37°C, 16 hours). The colonies formed on the agar were used as a random variant library.

[0103] Example 3 Screening of FpL_C3 amino acid substitutes (1) The random variant library (transformer) prepared in Example 2 or the transformant expressing FpL_C3 (serial number 1) prepared in Example 1 was inoculated into 250 µL of 2×YT liquid medium containing 50 μg / mL kanamycin (tryptone 1.6% (w / v), yeast extract 1% (w / v), sodium chloride 0.5% (w / v)) and cultured overnight at 37°C using a 96-well deep plate with shaking.

[0104] (2) After culturing, 5 µL of the culture medium from (1) was passaged and inoculated into 500 µL of 2×YT liquid medium containing 50 μg / mL kanamycin, 0.3% (w / v) glycine and 0.01 mM IPTG (Isopropyl-β-D-thiogalactopyranoside), and then further cultured overnight at 20 °C using a 96-well deep plate with shaking.

[0105] (3) After culturing, the culture supernatant obtained by centrifugation is mixed with an equal amount of 1.0M NaOH and treated with alkali at 38°C for 15 minutes.

[0106] (4) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method shown below. The residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment (3) by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment (3). (4-1) Human antibody (gamma globulin preparation, Institute of Chemistry and Serum Therapy) prepared at 10 μg / mL using Tris-Buffered Saline (TBS) was dispensed into each well of a 96-well microplate and fixed (4°C, 16 h). After fixation, the plate was blocked with 2% (w / v) skim milk (BD Biosciences) prepared using TBS. (4-2) After washing each well of the 96-well microplate with washing buffer (0.05M Tris buffer (pH 7.5) containing 0.15M NaCl and 0.05% (w / v) Tween 20 (trade name), also referred to as "washing buffer A"), add a solution containing immunoglobulin-binding proteins for evaluating antibody binding activity, and react the immunoglobulin-binding proteins with immobilized gamma globulin (30°C, 1 time). (4-3) After the reaction is complete, wash with washing buffer A, and add Anti-6-His Antibody (BETHYL LABORATORIES) diluted to 100 ng / mL at 100 µL / well for reaction (30 °C, 1 h). (4-4) After the reaction was complete, wash with washing buffer A, and add 50 µL of TMB peroxidase substrate (KPL) to each well. Then stop the reaction by adding 1 M phosphate at 50 µL per well, and measure the absorbance at 450 nm using a microplate reader (TECAN).

[0107] (5) Evaluate approximately 1,600 transformants and select those expressing immunoglobulin-binding proteins with improved alkali stability compared to the native type (without amino acid substitutions) FpL_C3 (Sequence No. 1).

[0108] (6) Cultivate the selected transformants and prepare expression vectors using the QIAprep Spin Miniprep kit (QIAGEN).

[0109] (7) For the sequence of the multinucleotide region encoding the immunoglobulin-binding protein inserted into the obtained expression vector, the nucleotide sequence was analyzed by the method described in Example 1 (5) to determine the amino acid substitution sites.

[0110] (8) The transformants expressing immunoglobulin-binding proteins selected in (5) were inoculated into 2 mL of 2×YT liquid medium containing 50 μg / mL kanamycin and cultured aerobically overnight at 37°C with shaking, thereby performing pre-culture.

[0111] (9) Inoculate 200µL of pre-culture solution into 20mL of 2×YT liquid medium containing 50μg / mL kanamycin and culture with shaking at 37℃.

[0112] (10) Two hours after the start of the culture, the culture temperature was changed to 20°C, and IPTG was added to a final concentration of 0.01mM. The culture was then aerobically shaken at 20°C overnight.

[0113] (11) After the culture was completed, the bacterial cells were collected by centrifugation and the protein extract was prepared using BugBuster protein extraction reagent (Merck).

[0114] (12) The antibody binding activity of immunoglobulin-binding proteins in the protein extract prepared in (11) was determined using the ELISA method described in (4).

[0115] (13) Dilute with TBS to ensure that the concentration of each protein is equal. Divide the diluent into two equal parts, add an equal amount of 0.2M NaOH (alkali treatment) to one part and an equal amount of TBS (untreated) to the other part, mix them, and then let stand at 25°C for 5 hours.

[0116] (14) After adding 4 times the volume of 1M Tris buffer (pH 7.0), the antibody binding activity of the protein solutions treated with alkali (treated with 0.1M NaOH at 25°C for 5 hours) and untreated with alkali was determined by the ELISA method described in (4). The residual activity was calculated by dividing the antibody binding activity of the alkali-treated immunoglobulin-binding protein by the antibody binding activity of the untreated immunoglobulin-binding protein, and the alkali stability was evaluated.

[0117] The results are shown in Table 2. It can be considered that immunoglobulin-binding proteins with at least one amino acid substitution in the amino acid sequence described in Serial No. 1, selected from P3S (this expression indicates that the amino acid residue equivalent to proline at position 3 of Serial No. 1 is replaced by serine, and the same applies below), E5V, E8G, I17F, E27G, A41T, N44S, E49V, N50S, N50Y, N50K, N50D, E52V, E52G, T54I, N62Y, and N65Y, have improved base stability compared to the native FpL_C3 (Serial No. 1).

[0118] Among them, the immunoglobulin-binding protein with amino acid substitutions of N50Y and E52G (Sequence No. 11, named FpL_C3 2a) was found to have significantly improved base stability (native FpL_C3: 40%, FpL_C32a: 94%). The immunoglobulin-binding protein with amino acid substitution of E52G alone (Sequence No. 16) did not show a significant improvement in base stability (native FpL_C3: 40%, Sequence No. 16: 48%). Therefore, it is speculated that the amino acid substitution that brings about the significant improvement in base stability is N50Y.

[0119] [Table 2]

[0120] Example 4: Preparation of FpL_C3 amino acid substituted aggregates (Part 1) (1) To produce an immunoglobulin-binding protein (Sequence No. 18, hereinafter also referred to as “FpL_C3KR”) by replacing all lysine residues of FpL_C3 (Sequence No. 1) with arginine residues.

[0121] (2) Further improvements in stability were sought by aggregating amino acid substitutions associated with the enhancement of alkaline stability of immunoglobulin-binding proteins, as identified in Example 2, onto FpL_C3KR (Sequence No. 18). Specifically, a protein (named FpL_C3KR 3a) was prepared by introducing amino acid substitutions of N50Y, E52G, and N62Y into FpL_C3KR.

[0122] Example 5: Preparation of FpL_C3 amino acid substituted aggregates (Part 2) (1) An immunoglobulin-binding protein FpL_C3KX (Sequence No. 20) is constructed in FpL_C3KR (Sequence No. 18) in which a portion of the arginine residues that were replaced by lysine residues are replaced by other amino acid residues (specifically, amino acid substitutions with K(R)7A (this expression indicates that the amino acid residue equivalent to lysine at position 7 of Sequence No. 1 is temporarily replaced by arginine and then further replaced by alanine, the same below), K(R)22Q and K(R)29I.

[0123] (2) Amino acid substitutions associated with improved alkaline stability of immunoglobulin-binding proteins, as identified in Example 2, were aggregated in FpL_C3KX (serial number 20). Specifically, a protein (named FpL_C3KX 3a) was prepared by introducing amino acid substitutions of N50Y, E52G, and N62Y into (r)FpL_C3KX.

[0124] Example 6: Evaluation of the base stability of FpL_C3 amino acid substituted aggregates (1) A protein extract was prepared by culturing a transformant expressing any one of the natural FpL_C3 (serial number 1) prepared in Example 1, the variant (amino acid substituted) immunoglobulin-binding proteins FpL_C3KR (serial number 18) and FpL_C3KR 3a (serial number 19) prepared in Example 4, and the variant immunoglobulin-binding proteins FpL_C3KX (serial number 20) and FpL_C3KX 3a (serial number 21) prepared in Example 5, using the same method as in Examples 3 (8) to (10).

[0125] (2) The antibody binding activity of the immunoglobulin-binding proteins in the protein extract prepared in (1) was determined using the ELISA method described in Example 3 (4).

[0126] (3) The concentration of NaOH used in the treatment was set to 0.4M (final concentration 0.2M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0127] The results are shown in Table 3. It can be concluded that immunoglobulin-binding proteins with all amino acid substitutions of N50Y, E52G, and N62Y (FpL_C3KR 3a and FpL_C3KX 3a) exhibit particularly improved basic stability compared to immunoglobulin-binding proteins without these substitutions (FpL_C3KR: 54%, FpL_C3KR 3a: 86%) (FpL_C3KX: 10%, FpL_C3KX 3a: 69%).

[0128] [Table 3]

[0129] Example 7 Screening of FpL_C3KX 3a amino acid substitutes (1) Using the same method as in Example 2, mutations were randomly introduced into the polynucleotide portion of the vector pET-FpL_C3KX3a, which was obtained by inserting the polynucleotide encoding FpL_C3KX3a (sequence number 21) into plasmid pET-26b and capable of expressing FpL_C3KX3a, to create a library via error-prone PCR. The average mutation introduction rate was 1.7%.

[0130] (2) Transformers that express FpL_C3KX 3a (serial number 21) and the library of random variants prepared in (1) were cultured by means of pET-FpL_C3KX 3a to Escherichia coli BL21 (DE3) strain by the same method as in Examples 3 (1) to (2).

[0131] (3) After culturing, the culture supernatant obtained by centrifugation is mixed with an equal amount of 1.0M NaOH and treated with alkali at 42°C for 30 minutes.

[0132] (4) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method described in Example 3 (4), and the residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment.

[0133] (5) Evaluate approximately 1,400 transformants and select those expressing immunoglobulin-binding proteins with improved alkali stability compared to FpL_C3KX3a (serial number 21).

[0134] (6) Cultivate the selected transformant or the transformant expressing FpL_C3KX 3a (serial number 21) and prepare the expression vector using the QIAprepSpin Miniprep kit (QIAGEN).

[0135] (7) The sequence of the multinucleotide region encoding the immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid variation sites.

[0136] (8) The transformants expressing immunoglobulin-binding proteins selected in (1) were cultured by the same method as in Examples 3 (8) to (10), and the protein extract was prepared by the same method as in Example 3 (11).

[0137] (9) The antibody binding activity of the immunoglobulin-binding proteins in the protein extract prepared in (8) was determined using the ELISA method described in Example 3 (4).

[0138] (10) The concentration of NaOH used in the treatment was set to 0.4M (final concentration 0.2M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0139] The results are shown in Table 4. It can be concluded that immunoglobulin-binding proteins with at least one amino acid substitution selected from E4K, E27R and T36A in the amino acid sequence described in Serial No. 21 have improved base stability compared to FpL_C3KX 3a (Serial No. 21), which has improved base stability compared to the native type FpL_C3 (Serial No. 1).

[0140] [Table 4]

[0141] Example 8 Evaluation of immunoglobulin κ light chain binding of immunoglobulin-binding proteins (Part 1) The effect of amino acid substitution on the binding activity of human immunoglobulin κ light chain was evaluated by measuring the immunoglobulin κ light chain binding activity of variant immunoglobulin-binding proteins using the ELISA method shown below.

[0142] (1) Transformations expressing any one of the following proteins were cultured using the same method as in Examples 3 (8) to (10): FpL_C3KR (serial number 18) prepared in Example 4, FpL_C3KX 3a (serial number 21) prepared in Example 5, and an immunoglobulin-binding protein (serial number 26, hereinafter also referred to as "FpL_C3KX 4h") into which E49D amino acids were introduced in FpL_C3KX 3a obtained in Example 7. Protein extracts were prepared using the same method as in Example 3 (11). The extracts were centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0143] (2) Add the clarified supernatant from (1) to a column filled with Ni-NTA Sepharose 6 Fast Flow (Cytiva) pre-equilibrated with 0.05M Tris buffer (pH 7.5) containing 0.5M NaCl and 0.02M imidazole (hereinafter referred to as washing buffer B), wash with 10 times the amount of the carrier filled in the column with washing buffer B, and then pass through 0.05M Tris buffer (pH 7.5) containing 0.5M NaCl and 0.5M imidazole, thereby recovering the fraction equivalent to immunoglobulin-binding proteins.

[0144] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the various variant immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE (SDS-polyacrylamide electrophoresis) was performed to confirm that the various variant immunoglobulin-binding proteins were purified to good purity.

[0145] (4) The binding activity of human immunoglobulin κ light chain of variant immunoglobulin-binding protein was determined by the ELISA method shown below, and the effect of amino acid substitution on the binding activity of human immunoglobulin κ light chain was evaluated. (4-1) The monoclonal antibody solution prepared using TBS at a concentration of 10 μg / mL was dispensed into the wells of a 96-well microplate and fixed (4°C, 16 hours). The polyclonal antibody used was any one of the following: Herceptin (manufactured by Chugai Pharmaceutical Co., Ltd.), which contains human κ light chain subgroup 1 (Vκ1), Human PDGF R alpha antibody containing human κ light chain subgroup 3 (Vκ3) (manufactured by R&D Systems) Human CTLA4 antibody containing human κ light chain subgroup 3 (Vκ3) (manufactured by R&D Systems), and Human PCSK9 antibody containing human κ light chain subgroup 4 (Vκ4) (manufactured by R&D Systems). After immobilization, the cells were blocked using a 2% (w / v) skim milk (manufactured by BD Corporation) prepared using TBS. (4-2) After washing the wells of the 96-well microplate with washing buffer A (0.15M NaCl and 0.05% (w / v) Tween 20 (trade name) in 0.05M Tris buffer (pH 7.5)), add a solution diluted to 50 μg / mL containing immunoglobulin-binding proteins for evaluating antibody binding activity to allow the immunoglobulin-binding proteins and immobilized polyclonal antibodies to react (30°C, 1 hour). (4-3) After the reaction is complete, wash with washing buffer A, and add Anti-6-His Antibody (BETHYL LABORATORIES) diluted to 100 ng / mL at 100 µL / well for reaction (30 °C, 1 h). (4-4) After the reaction was complete, wash with washing buffer A, and add 50 µL of TMB peroxidase substrate (KPL) to each well. Then stop the reaction by adding 1 M phosphate at 50 µL per well, and measure the absorbance at 450 nm using a microplate reader (TECAN). (4-5) Calculate the difference in absorbance between the wells with immobilized polyclonal antibodies and those without. Use this calculated absorbance to evaluate the binding affinity of the monoclonal antibody to variant immunoglobulin-binding proteins. The difference in absorbance between the monoclonal antibodies indicates the binding affinity to different human κ light chains. A large deviation in this value for each polyclonal antibody indicates a decreased binding affinity to a specific human κ light chain subgroup.

[0146] The results are shown in Table 5. FpL_C3KX_4h (serial number 26) can be considered to bind to the human immunoglobulin κ light chain independently of subgroups due to the small deviation among the monoclonal antibodies. On the other hand, FpL_C3KR (serial number 18) and FpL_C3KX3a (serial number 21) showed particularly reduced binding affinity to human Vκ3. Based on the above results, it can be concluded that introducing amino acid substitutions of E49D can impart binding affinity to Vκ3.

[0147] [Table 5]

[0148] Example 9 Screening of FpL_C3KX 4h amino acid substitutes Using the same method as in Example 2, a library was constructed by randomly introducing mutations into the multinucleotide moiety (Sequence No. 27) encoding an immunoglobulin-binding protein in the vector pET-FpL_C3KX 4h (Sequence No. 26) expressing FpL_C3KX 4h obtained in Example 7 via error-prone PCR. The average mutation introduction rate was 1.7%.

[0149] (2) After culturing the random variant library (transformer) prepared in (1) by the same method as in Examples 3 (1) to (2), the culture supernatant obtained by centrifugation was subjected to the alkali treatment described in Example 19 (2).

[0150] (3) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method described in Example 3 (4), and the residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment.

[0151] (4) Evaluate approximately 900 transformants and select those expressing immunoglobulin-binding proteins with increased alkali stability compared to those expressing immunoglobulin-binding proteins with increased alkali stability (FpL_C3KX 4h).

[0152] (5) Cultivate the selected transformants and prepare expression vectors using the QIAprep Spin Miniprep kit (QIAGEN).

[0153] (6) The sequence of the multinucleotide region encoding immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid variation sites.

[0154] (7) The transformant expressing the immunoglobulin-binding protein selected in (6) or the transformant expressing FpL_C3KX 4h (serial number 26) obtained in Example 7 was cultured in the same manner as in Examples 3 (8) to (10), and a protein extract was prepared in the same manner as in Example 3 (11). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0155] (8) The supernatant clarified in (7) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0156] (9) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0157] (10) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0158] The results are shown in Table 6. It can be concluded that immunoglobulin-binding proteins with at least one amino acid substitution selected from E9V, Y53F, T54M and A69T in the amino acid sequence described in Serial No. 26 have improved base stability compared to FpL_C3KX 4h (Serial No. 26), which has improved base stability compared to the native type FpL_C3 (Serial No. 1).

[0159] Furthermore, the purification yield was compared when cultured in 20 mL of medium, and the results confirmed that the protein purification yield was significantly improved by introducing amino acid substitutions of K(Q)22R. The immunoglobulin-binding protein for which K(Q)22R amino acid substitutions were introduced into FpL_C3KX 4h is named FpL_C3KX 4i (serial number 29).

[0160] [Table 6]

[0161] Example 10: Preparation of FpL_C3KX 4i amino acid substituted aggregates Further improvements in stability were sought by aggregating amino acid substitutions associated with enhanced alkaline stability of immunoglobulin-binding proteins, as identified in Example 9, into FpL_C3KX 4i (Sequence No. 29). Specifically, a protein (Sequence No. 33, named FpL_C3KX 5e) was designed and fabricated by introducing the amino acid substitution Y53F into FpL_C3KX 4i.

[0162] Example 11: Evaluation of the base stability of FpL_C3KX 4h amino acid substitutes (1) Transformants expressing any one of FpL_C3KX4h (serial number 26) obtained in Example 7, FpL_C3KX4i (serial number 29) obtained in Example 9, and FpL_C3KX5e (serial number 33) prepared in Example 10 were cultured using the same method as in Examples 3 (8) to (10). Protein extracts were prepared using the same method as in Example 3 (11). The extracts were centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0163] (2) The supernatant clarified in (1) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0164] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0165] (4) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment temperature was set to 55°C, and the treatment time was set to 15 minutes. Otherwise, the alkali treatment was carried out in the same way as in Example 3 (13), and the residual activity was calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0166] The results are shown in Table 7. It can be considered that the FpL_C3KX 5e (serial number 33) prepared in Example 10 has improved alkali stability compared to FpL_C3KX 4h (serial number 26) and FpL_C3KX4i (serial number 29), which have improved alkali stability compared to the natural FpL_C3 (serial number 1) (FpL_C3KX 4h: 34%, FpL_C3KX 4i: 48%, FpL_C3KX5e: 73%).

[0167] [Table 7]

[0168] Example 12 Screening of FpL_C3KX 5e amino acid substitutes (1) Using the same method as in Example 2, a library was prepared by randomly introducing a mutation into the polynucleotide portion (Sequence No. 34) encoding an immunoglobulin-binding protein in the vector pET-FpL_C3KX 5e (Sequence No. 33) expressing FpL_C3KX 5e obtained in Example 10 via error-prone PCR. The average mutation introduction rate was 1.9%.

[0169] (2) After culturing the random variant library (transformer) prepared in (1) by the same method as in Examples 3 (1) to (2), the culture supernatant obtained by centrifugation was subjected to the alkali treatment described in Example 19 (2).

[0170] (3) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method described in Example 3 (4), and the residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment.

[0171] (4) Evaluate approximately 900 transformants and select those expressing immunoglobulin-binding proteins with increased alkali stability compared to the alkali-stable immunoglobulin-binding protein (FpL_C3KX 5e).

[0172] (5) Cultivate the selected transformants and prepare expression vectors using the QIAprep Spin Miniprep kit (QIAGEN).

[0173] (6) The sequence of the multinucleotide region encoding immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid variation sites.

[0174] (7) The protein extract was prepared by culturing the transformant expressing the immunoglobulin-binding protein selected in (4) or the transformant expressing FpL_C3KX 5e (serial number 33) prepared in Example 10 using the same method as in Examples 3 (8) to (10). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0175] (8) The supernatant clarified in (7) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0176] (9) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0177] (10) The concentration of NaOH used in the treatment was set to 2.0M (final concentration 1.0M), the treatment time was set to 17 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0178] The results are shown in Table 8. It can be considered that immunoglobulin-binding proteins with at least one amino acid substitution selected from E1V, E4G, G21R and E(G)52D in the amino acid sequence described in Serial No. 33 have improved base stability compared to FpL_C3KX 5e (Serial No. 33), which has improved base stability compared to the native type FpL_C3 (Serial No. 1).

[0179] [Table 8]

[0180] Example 13 Evaluation of immunoglobulin κ light chain binding of immunoglobulin-binding proteins (Part 2) The effect of introducing amino acid substitutions of P6S on human immunoglobulin κ light chain binding activity was evaluated. Specifically, an immunoglobulin-binding protein (named FpL_C3KX 5e_P6S, SEQ ID NO. 40) with P6S amino acid substitutions introduced into FpL_C3KX 5e (SEQ ID NO. 33) was constructed and evaluated.

[0181] (1) Transformants expressing either FpL_C3KX_5e_P6S (serial number 40) or FpL_C3KX_5e (serial number 33) were cultured using the same method as in Examples 3 (8) to (10), and a protein extract was prepared using the same method as in Example 3 (11). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0182] (2) The supernatant clarified in (1) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0183] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the various variant immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the various variant immunoglobulin-binding proteins were purified to good purity.

[0184] (4) The binding activity of human immunoglobulin κ light chain of immunoglobulin-binding proteins was determined using the same method as in Example 8 (4), and the effect of amino acid substitution on the binding activity of human immunoglobulin κ light chain was evaluated. The monoclonal antibody used in the binding activity evaluation was any one of the following: Herceptin (manufactured by Chugai Pharmaceutical Co., Ltd.), which contains human κ light chain subgroup 1 (Vκ1), and Human CTLA4 antibody containing human κ light chain subgroup 3 (Vκ3) (manufactured by R&D Systems).

[0185] Reference Example 1 The effect of introducing amino acid substitutions of K38E on human immunoglobulin κ light chain binding activity was evaluated. Specifically, an immunoglobulin-binding protein (Sequence No. 41, named FpL_C3KR_K38E) with amino acid substitutions of K(R)38E introduced into FpL_C3KR (Sequence No. 18) was constructed and evaluated.

[0186] (1) Transformants expressing either FpL_C3KR_K38E (serial number 41) or FpL_C3KR (serial number 18) prepared in Example 4 were cultured using the same method as in Examples 3 (8) to (10). Protein extracts were prepared using the same method as in Example 3 (11). The extracts were centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0187] (2) The supernatant clarified in (1) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0188] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the various variant immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the various variant immunoglobulin-binding proteins were purified to good purity.

[0189] (4) The binding activity of human immunoglobulin κ light chain of immunoglobulin-binding protein was determined by the same method as in Example 13 (4), and the effect of amino acid substitution on the binding activity of human immunoglobulin κ light chain was evaluated.

[0190] The results of Example 13 and Reference Example 1 are shown together in Table 9. It can be seen that by introducing amino acid substitutions of P6S (Example 13) or K(R)38E (Reference Example 1), binding affinity with Vκ3 can be acquired.

[0191] [Table 9]

[0192] Example 14: Preparation of FpL_C3KX 5e amino acid substituted aggregates The amino acid substitutions that impart binding affinity to Vκ3 and the amino acid substitutions related to improved alkaline stability of immunoglobulin-binding proteins, as identified in Example 27, were aggregated in FpL_C3KX 5e. Specifically, a protein (serial number 42, named FpL_C3KX 7d) was designed and fabricated by introducing amino acid substitutions of E4G, P6S, K(R)38E, and E(G)52D into FpL_C3KX 5e.

[0193] Example 15: Evaluation of the base stability of FpL_C3KX 5e amino acid substituted aggregates (1) Transformants expressing either FpL_C3KX5e (serial number 33) prepared in Example 10 or FpL_C3KX 7d (serial number 42) prepared in Example 14 were cultured using the same method as in Examples 3 (8) to (10), and a protein extract was prepared using the method described in Example 3 (11). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0194] (2) The supernatant clarified in (1) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0195] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0196] (4) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0197] The results are shown in Table 10. It can be considered that the FpL_C3KX 7d (serial number 42) evaluated in this embodiment has improved alkali stability compared to FpL_C3KX 5e (serial number 33), which has improved alkali stability compared to the natural FpL_C3 (serial number 1).

[0198] [Table 10]

[0199] Example 16 Screening of FpL_C3KX 7d amino acid substituents (Part 1) (1) Using the same method as in Example 2, a library was prepared by randomly introducing a mutation into the multinucleotide portion (sequence number 43) encoding an immunoglobulin-binding protein in the vector pET-FpL_C3KX 7d (sequence number 42) expressing FpL_C3KX 7d prepared in Example 14 via error-prone PCR. The average mutation introduction rate was 1.9%.

[0200] (2) Transformers capable of expressing FpL_C3KX 7d and libraries of random variants prepared in (1) were cultured using the same method as in Examples 3 (1) to (2).

[0201] (3) After culturing, the culture supernatant obtained by centrifugation is mixed with an equal amount of 1.0M NaOH and treated with alkali at 58°C for 30 minutes.

[0202] (4) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method described in Example 3 (4), and the residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment.

[0203] (5) Evaluate approximately 1,000 transformants and select those expressing immunoglobulin-binding proteins with improved alkali stability compared to FpL_C3KX7d (serial number 42).

[0204] (6) Cultivate the selected transformants and prepare expression vectors using the QIAprep Spin Miniprep kit (QIAGEN).

[0205] (7) The sequence of the multinucleotide region encoding the immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid variation sites.

[0206] (8) The transformant expressing the immunoglobulin-binding protein selected in (5) or the transformant expressing FpL_C3KX 7d (serial number 42) was cultured in the same manner as in Examples 3 (8) to (10), and a protein extract was prepared in the same manner as in Example 3 (11). After centrifugation of the extract, the supernatant was clarified by passing it through a filter.

[0207] (9) The supernatant clarified in (8) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0208] (10) The absorbance of the recovered fraction was determined using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0209] (11) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0210] The results are shown in Table 11. It can be considered that immunoglobulin-binding proteins with at least one amino acid substitution selected from E8G, K(I)29F, E33V and A69V in the amino acid sequence described in Serial No. 42 have improved base stability compared to FpL_C3KX 7d (Serial No. 42), which has improved base stability compared to the native type FpL_C3 (Serial No. 1).

[0211] [Table 11]

[0212] Example 17: Importing Mutants into FpL_C3KX 7d and Creating a Library (1) Using the vector pET-FpL_C3KX 7d, which is capable of expressing FpL_C3KX 7d prepared in Example 14, as a template, a library of saturated substitutions targeting amino acid residues at positions 23, 41, and 52 was prepared. Primers were designed according to the 22c-Trick method (Sabrina Kille et al., ACS Synthetic Biology 2013; 2: 83-92) to provide appropriate mixed bases at the mutation introduction site and to enable reverse PCR centered on the mutation introduction site (sequence numbers 47 to 52, 54 to 59, and 62 to 67). For each substitution site, six primers were mixed according to the compositions in Tables 12 and 13 to prepare a PCR primer mix (Forward / Reverse primer mix).

[0213] [Table 12]

[0214] [Table 13]

[0215] (2) pET-FpL_C3KX 7d was used as template DNA and site-specific mutations were introduced by reverse PCR. Reverse PCR was performed as follows: After preparing the reaction solution with the composition shown in Table 14, the reaction was performed for 30 cycles. The reaction solution was heat-treated at 98°C for 2 minutes, and the first step was heat-treated at 95°C for 10 seconds, the second step was heat-treated at 50°C for 5 seconds, and the third step was heat-treated at 72°C for 6 minutes, which constituted one cycle. Finally, the reaction solution was heat-treated at 72°C for 7 minutes.

[0216] [Table 14]

[0217] (3) After purifying the obtained PCR product, Escherichia coli BL21 (DE3) strain was transformed with the reaction solution and cultured on LB agar containing 50 μg / mL kanamycin (37°C, 16 hours). The colonies formed on the agar were used as site-specific variant libraries.

[0218] Example 18 Screening of FpL_C3KX 7d amino acid substituents (Part 2) (1) After culturing the transformant that can express FpL_C3KX 7d and the site-specific amino acid substitute library (transformant) prepared in Example 17 by the same method as in Examples 3 (1) to (2), the culture supernatant obtained by centrifugation was subjected to the alkali treatment described in Example 16 (3).

[0219] (2) The antibody binding activity of immunoglobulin-binding proteins under alkaline treatment was determined by the ELISA method described in Example 3 (4), and the residual activity was calculated by dividing the antibody binding activity of immunoglobulin-binding proteins under alkaline treatment by the antibody binding activity of immunoglobulin-binding proteins without alkaline treatment.

[0220] (3) Evaluate about 300 transformants and select transformants that express immunoglobulin-binding proteins with improved alkali stability compared to FpL_C3KX7d (serial number 42).

[0221] (4) Cultivate the selected transformants and prepare expression vectors using the QIAprep Spin Miniprep kit (QIAGEN).

[0222] (5) The sequence of the multinucleotide region encoding immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid variation sites.

[0223] (6) The transformant expressing the immunoglobulin-binding protein selected in (3) or the transformant expressing FpL_C3KX 7d (serial number 42) prepared in Example 14 was cultured by the same method as in Examples 3 (8) to (10), and a protein extract was prepared by the same method as in Example 3 (11). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0224] (7) The supernatant clarified in (6) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0225] (8) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0226] (9) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0227] The results are shown in Table 15. It can be considered that immunoglobulin-binding proteins with at least the amino acid substitutions of I23R, A41I, A41Y, E(G,D)52L (this expression indicates that the amino acid residue equivalent to glutamic acid at position 7 of sequence number 1 is temporarily substituted with glycine, then with aspartic acid, and then with leucine, and so on) and E(G,D)52V in the amino acid sequence described in sequence number 42 have improved base stability compared to FpL_C3KX 7d (sequence number 42), which has improved base stability compared to the native type FpL_C3 (sequence number 1).

[0228] [Table 15]

[0229] Example 19: Preparation of FpL_C3KX 7d amino acid substituted aggregates Further improvements in base stability were sought by aggregating amino acid substitutions associated with enhanced base stability of immunoglobulin-binding proteins, as identified in Examples 16 and 18, into FpL_C3KX 7d (Sequence No. 42). Specifically, a protein (Sequence No. 70, named FpL_C3KX 8b) was designed and fabricated by introducing amino acid substitutions of E(G,D)52L and A69V into FpL_C3KX 7d.

[0230] Example 20: Evaluation of the basic stability of FpL_C3KX 7d amino acid substituted aggregates (1) Transformants expressing either FpL_C3KX7d_A69V (serial number 44) obtained in Example 16 or FpL_C3KX8b (serial number 70) prepared in Example 18 were cultured using the same method as in Examples 3 (8) to (10), and a protein extract was prepared using the method described in Example 3 (11). The extract was centrifuged, and the resulting supernatant was clarified by passing it through a filter.

[0231] (2) The supernatant clarified in (1) was purified by the method described in Example 8 (2) to recover a fraction equivalent to immunoglobulin-binding proteins.

[0232] (3) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0233] (4) The concentration of NaOH used in the treatment was set to 1.0M (final concentration 0.5M), the treatment time was set to 15 hours, and the alkali treatment was carried out in the same way as in Example 3 (13). The residual activity was then calculated and the alkali stability was evaluated in the same way as in Example 3 (14).

[0234] The results are shown in Table 16. It can be considered that FpL_C3KX 8b (serial number 70) has improved alkali stability compared to FpL_C3KX 7d_A69V (serial number 44), which has improved alkali stability compared to the natural FpL_C3 (serial number 1).

[0235] [Table 16]

[0236] Comparative Example 1: Preparation of FpL_C3 (1) The recombinant Escherichia coli that expressed FpL_C3 obtained in Example 1 was inoculated into 20 mL of 2×YT liquid medium containing 50 μg / mL kanamycin (1.6% tryptone (w / v), 1% yeast extract (w / v), 0.5% sodium chloride (w / v)) and pre-cultured (30°C, 16 hours).

[0237] (2) Inoculate 10 mL of the pre-cultured medium into 1 L of 2×YT liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 2 to 3 hours, add 200 µL of 0.5 M IPTG and further culture at 20°C with shaking overnight.

[0238] (3) Centrifuge the culture medium after culturing and recover the cultured cells (8g wet weight). For the 2g wet weight of the cultured cells, use BugBuster protein extraction reagent (Merck Millipore) to lyse them. Clarify the supernatant obtained by centrifugation by passing it through a filter.

[0239] (4) Add the clarified supernatant from (3) to a column packed with Ni Sepharose 6 Fast Flow (Cytiva) pre-equilibrated with 0.02M Tris-oat buffer (pH 7.5) containing 0.5M sodium chloride and 0.02M imidazole (hereinafter simply referred to as "equilibration buffer"). After washing with 10 times the volume of the equilibration buffer packed in the column, pass through 0.02M Tris-oat buffer (pH 7.5) containing 0.5M sodium chloride and 0.5M imidazole to recover the fraction equivalent to immunoglobulin-binding proteins.

[0240] (5) The absorbance of the recovered fraction was measured by spectrophotometer to quantify the amount of protein contained in the fraction. In addition, SDS-PAGE was performed to confirm that the immunoglobulin-binding protein was purified to a good purity.

[0241] Example 21 Evaluation of immunoglobulin κ light chain binding of immunoglobulin-binding proteins (Part 3) The immunoglobulin κ light chain binding activity of FpL_C3 prepared in Comparative Example 1 and FpL_C3KX 8b prepared in Example 19 was determined using the same method as in Example 8 (4) to evaluate the effect of amino acid substitution on the human immunoglobulin κ light chain binding activity. The monoclonal antibody used in the binding activity evaluation was any one of the following: Humira (manufactured by Eisai) contains human κ light chain subgroup 1 (Vκ1). Human PDGF R alpha antibody containing human κ light chain subgroup 3 (Vκ3) (manufactured by R&D Systems), and Human PCSK9 antibody containing human κ light chain subgroup 4 (Vκ4) (manufactured by R&D Systems).

[0242] The results are shown in Table 17. It can be concluded that FpL_C3 (Sequence No. 1) can bind to the human immunoglobulin κ light chain independently of subgroups because it shows the same degree of color development in all monoclonal antibodies. On the other hand, FpL_C3KX8b (Sequence No. 70) exhibits reduced binding affinity to Vκ3 and Vκ4. Based on the above results, it can be concluded that FpL_C3KX8b has improved selectivity for Vκ1 compared to FpL_C3.

[0243] [Table 17]

[0244] Example 22 Screening of FpL_C3KX8b amino acid substitutes (1) Using the vector pET-FpL_C3KX 8b, which is capable of expressing FpL_C3KX 8b prepared in Example 19, as a template, a library of saturated substitutions targeting amino acid residues at positions 22, 38, 42, and 44 was prepared. The primers were designed in the same manner as in Example 17 (1) according to the 22c-Trick method, with appropriate mixed bases at the mutation introduction site, enabling reverse PCR centered on the mutation introduction site (serial numbers 71 to 94). For each substitution site, six primers were mixed in a group according to the compositions in Tables 12 and 18 to prepare a PCR primer mix (Forward / Reverse primer mix).

[0245] [Table 18]

[0246] (2) pET-FpL_C3KX 8b prepared in Example 19 was used as template DNA, and site-specific mutations were introduced by reverse PCR in the same manner as in Example 17 (2).

[0247] (3) After purifying the obtained PCR product, use the purified product to transform Escherichia coli BL21(DE3) strain, and culture it in LB agar medium containing 50 μg / mL kanamycin (37℃, 16 hours). The colonies formed on the agar plate are used as saturated substitute libraries.

[0248] (4) The transformant that can express FpL_C3KX 8b prepared in Example 19 and the saturated substitute library (transformant) prepared in (3) were inoculated into 250 µL of 2×YT liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking in a 96-well deep plate.

[0249] (5) After culturing, 5 µL of the culture medium was subcultured and inoculated into 500 µL of 2×YT liquid medium containing 50 μg / mL kanamycin, 0.3% (w / v) glycine and 0.01 mM IPTG, and then cultured overnight at 20 °C using a 96-well deep plate with shaking.

[0250] (6) After culturing, the culture supernatant obtained by centrifugation is mixed with an equal amount of TBS.

[0251] (7) Using the same method as in Example 21, the antibody binding activity of the κ light chain relative to different subgroups was evaluated, and the transformant with fewer chromogenic wells immobilized with Vκ3 or Vκ4 compared to FpL_C3KX 8b was selected.

[0252] (8) Evaluate approximately 200 transformants and select those expressing immunoglobulin-binding proteins with reduced binding affinity to Vκ3 or Vκ4 compared to FpL_C3KX8b.

[0253] (9) Cultivate the transformants selected in (8) and prepare expression vectors using QIAprep SpinMiniprep kit (QIAGEN).

[0254] (10) The sequence of the multinucleotide region encoding immunoglobulin-binding protein inserted in the obtained expression vector was analyzed by the method described in Example 1 (5) to determine the amino acid substitution sites.

[0255] (11) The transformant expressing immunoglobulin-binding protein selected in (10) was cultured by the same method as in Examples 3 (8) to (10), and the protein extract was purified by the same method as in Examples 8 (1) to (2) to recover a fraction equivalent to the immunoglobulin-binding protein.

[0256] (12) The absorbance of the recovered fraction was measured using a spectrophotometer to quantify the immunoglobulin-binding proteins contained in the fraction. In addition, SDS-PAGE was performed to confirm that the selected immunoglobulin-binding proteins were purified to good purity.

[0257] (13) The binding activity of the immunoglobulin-binding protein prepared in (12) to the monoclonal antibodies of each subgroup was determined by the same method as in Example 21.

[0258] The results are shown in Table 19. It was confirmed that immunoglobulin-binding proteins with at least one amino acid substitution selected from K(R)22E, K(R,E)38P, K(R,E)38D, Y42F, Y42H, Y42W, and N44P in the amino acid sequence described in Serial No. 1 exhibited improved selectivity for Vκ1 compared to FpL_C3KX 8b (Serial No. 70), which showed increased selectivity for Vκ1 compared to the native FpL_C3. Specifically, immunoglobulin-binding proteins with amino acid substitutions of K(R,E)38D, Y42F, or Y42H showed significantly reduced binding to both Vκ3 and Vκ4, indicating a further improvement in selectivity for Vκ1.

[0259] [Table 19]

[0260] Example 23: Preparation of FpL_C3KX 8b amino acid substituted aggregates Further upskilling of selectivity was attempted by aggregating the amino acid substitutions associated with increased selectivity for Vκ1, as identified in Example 22, into FpL_C3KX 8b (Sequence No. 70). Specifically, a protein (Sequence No. 102, named FpL_C3KX 8b_K38D,Y42H) was designed to introduce amino acid substitutions of K(R,E)38D and Y42H into FpL_C3KX 8b.

[0261] (1) By introducing variants through reverse PCR, polynucleotides encoding FpL_C3KX8b_K38D and Y42H were prepared. Reverse PCR was performed as follows: An expression vector containing the polynucleotide encoding FpL_C3KX8b_Y42H (sequence number 103) was used as template DNA. After preparing the reaction solution with the composition shown in Table 20, the reaction was performed for 30 cycles. The reaction solution was heat-treated at 98°C for 2 minutes. One cycle consisted of the first step of heat treatment at 95°C for 10 seconds, the second step of heat treatment at 50°C for 5 seconds, and the third step of heat treatment at 72°C for 1 minute. Finally, the reaction solution was heat-treated at 72°C for 1 minute.

[0262] [Table 20]

[0263] (2) The PCR product obtained in (1) was used to transform Escherichia coli BL21 (DE3) strain and cultured on LB (Luria-Bertani) plates containing 50 μg / mL kanamycin (37°C, 16 hours).

[0264] (3) After selecting the transformant (recombinant E. coli) obtained in (2), culture and plasmid purification were carried out in the same way as in Example 1 (4) to obtain a vector (named pET-FpL_C3KX 8b_E38D, Y42H) that can express FpL_C3KX 8b_E38D, Y42H.

[0265] (4) For the polynucleotides encoding FpL_C3KX8b_E38D and Y42H and their surrounding regions in the expression vectors pET-FpL_C3KX8b_E38D and Y42H obtained in (3), sequence analysis was performed using the same method as in Example 1 (5).

[0266] The sequence analysis confirmed that a polynucleotide encoding FpL_C3KX8b_E38D,Y42H (sequence number 102) was inserted into the expression vector pET-FpL_C3KX8b_E38D,Y42H (sequence number 103).

[0267] (5) FpL_C3KX8b_E38D and Y42H were prepared from the transformant obtained in (2) by the same method as in Comparative Example 1.

[0268] (6) The binding activity of the immunoglobulin-binding protein prepared in (5) to the monoclonal antibodies of each subgroup was determined by the same method as in Example 21.

[0269] The results are shown in Table 21. Compared with FpL_C3KX 8b (serial number 70), which has increased selectivity for Vκ1 compared with the natural type FpL_C3, FpL_C3KX8b_E38D and Y42H (serial number 102) showed decreased binding to Vκ3 and Vκ4. As a result, the selectivity for Vκ1 was significantly improved.

[0270] [Table 21] Industrial availability

[0271] The protein of the present invention is characterized by comprising an amino acid sequence containing an immunoglobulin-binding domain of protein L derived from the genus *Fingoldii*, wherein at least one amino acid substitution in the amino acid sequence enhances the binding selectivity to antibodies having a specific light chain, and the protein possesses immunoglobulin-binding activity. The immunoglobulin adsorbent of the present invention, comprising an insoluble carrier and the protein of the present invention immobilized on the insoluble carrier, can achieve the effect of purifying and separating bispecific antibodies that cannot be separated by conventional immunoglobulin adsorbents.

Claims

1. A protein comprising an amino acid sequence of an immunoglobulin-binding domain of protein L derived from Fingoldii bacteria, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5) below, and the protein has immunoglobulin-binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline.

2. The protein according to claim 1, wherein it is the protein described in any one of (a) to (c): (a) A protein comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence has at least one or more amino acid substitutions selected from (1) to (5) below, and the protein has immunoglobulin binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline; (b) A protein comprising the amino acid sequence described in sequence number 1, wherein the amino acid sequence comprises at least one amino acid substitution selected from (1) to (5), and in addition to (1) to (5), it comprises substitution, deletion, insertion, and / or addition of one or more amino acid residues at one or more positions, and the protein has immunoglobulin binding activity. (c) A protein comprising an amino acid sequence having more than 70% homology with the amino acid sequence or a portion thereof described in sequence number 1, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5), and the protein has immunoglobulin binding activity.

3. The protein according to claim 1, further comprising at least one amino acid substitution selected from (6) to (11): (6) The amino acid residue equivalent to asparagine at position 50 of sequence number 1 is replaced with tyrosine. (7) The amino acid residue equivalent to glutamic acid at position 4 of sequence number 1 is replaced with glycine. (8) The amino acid residue equivalent to lysine at position 7 of sequence number 1 is replaced with alanine. (9) The amino acid residue equivalent to glutamic acid at position 52 of sequence number 1 is replaced with leucine. (10) The amino acid residue equivalent to tyrosine at position 53 of sequence number 1 is replaced with phenylalanine. (11) The amino acid residue equivalent to alanine at position 69 of sequence number 1 is replaced with valine.

4. The protein according to claim 1, further comprising at least the following amino acid substitutions (6) and one or more amino acid substitutions selected from (7) to (11): (6) The amino acid residue equivalent to asparagine at position 50 of sequence number 1 is replaced with tyrosine. (7) The amino acid residue equivalent to glutamic acid at position 4 of sequence number 1 is replaced with glycine. (8) The amino acid residue equivalent to lysine at position 7 of sequence number 1 is replaced with alanine. (9) The amino acid residue equivalent to glutamic acid at position 52 of sequence number 1 is replaced with leucine. (10) The amino acid residue equivalent to tyrosine at position 53 of sequence number 1 is replaced with phenylalanine. (11) The amino acid residue equivalent to alanine at position 69 of sequence number 1 is replaced with valine.

5. The protein according to claim 1, further comprising at least the following (12) amino acid substitutions: (12) The amino acid residue equivalent to lysine at position 29 of sequence number 1 is replaced with isoleucine.

6. The protein according to claim 4, wherein it is any one of the proteins described in (d) to (f): (d) A protein comprising the amino acid sequence described in sequence number 70, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5) below, and the protein has immunoglobulin binding activity: (1) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with histidine. (2) The amino acid residue equivalent to lysine at position 22 of sequence number 1 is replaced with glutamic acid. (3) The amino acid residue equivalent to lysine at position 38 of sequence number 1 is replaced with proline or aspartic acid. (4) The amino acid residue equivalent to tyrosine at position 42 of sequence number 1 is replaced with tryptophan or phenylalanine. (5) The amino acid residue equivalent to asparagine at position 44 of sequence number 1 is replaced with proline; (e) A protein comprising the amino acid sequence described in sequence number 70, wherein the amino acid sequence comprises at least one amino acid substitution selected from (1) to (5), and in addition to (1) to (5), it comprises substitution, deletion, insertion, and / or addition of one or more amino acid residues at one or more positions, and the protein has immunoglobulin binding activity. (f) A protein comprising an amino acid sequence having more than 70% homology with the amino acid sequence or a portion thereof described in sequence number 70, wherein the amino acid sequence has at least one amino acid substitution selected from (1) to (5), and the protein has immunoglobulin binding activity.

7. A polynucleotide encoding the protein according to any one of claims 1 to 6.

8. An expression vector comprising the polynucleotide of claim 7.

9. An expression vector comprising a gene recombination host, said gene recombination host comprising (A) or (B): (A) A polynucleotide encoding the protein of any one of claims 1 to 6; and (B) A polynucleotide that encodes the protein of any one of claims 1 to 6.

10. The gene recombination host according to claim 9, wherein the host is Escherichia coli.

11. A method for manufacturing a protein with immunoglobulin-binding activity, comprising: The steps of culturing a gene recombinant host containing a polynucleotide encoding a protein according to any one of claims 1 to 6 or a gene recombinant host containing an expression vector containing the polynucleotide and expressing the protein therein; and the steps of recovering the expressed protein.

12. The manufacturing method according to claim 11, wherein the host is Escherichia coli.

13. An immunoglobulin adsorbent comprising an insoluble carrier and a protein of any one of claims 1 to 6 immobilized on the insoluble carrier.

14. A method for separating immunoglobulins contained in solution, comprising: The step of adding a solution containing immunoglobulins to a column filled with the adsorbent of claim 13 and allowing the immunoglobulins to be adsorbed onto the adsorbent; The process of eluting the immunoglobulins adsorbed on the adsorbent.

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