Preservation solution for immunoglobulin-binding protein

Abstract

To provide a preservation solution that enhances the stability of a polypeptide, comprising at least an immunoglobulin-binding domain of Protein L (FpL) from a Finegoldia bacterium.SOLUTION: The foregoing problem is solved by incorporating 0.03 mol / L or more and 3.0 mol / L or less of a sugar into the preservation solution.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a preservation solution for immunoglobulin-binding proteins. In particular, it relates to a preservation solution that improves the stability of a polypeptide containing at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia.

Background Art

[0002] Antibody drugs are drugs that utilize antibodies (immunoglobulins), which are molecules responsible for the immune function in vivo. Antibody drugs bind to target molecules with high specificity and affinity due to the diversity of the variable regions of antibodies. Therefore, antibody drugs have few side effects, and in recent years, the range of applicable diseases has been expanding, leading to a rapid expansion of the market.

[0003] The production of antibody drugs includes a culture process and a purification process. In the culture process, modifications of antibody-producing cells and optimization of culture conditions are carried out to improve productivity. In the purification process, affinity chromatography is employed as a crude purification step, followed by intermediate purification, final purification, and virus removal, and then formulated.

[0004] In the purification process, an affinity carrier that specifically recognizes antibody molecules is used. As the ligand protein used in the carrier, Protein A derived from bacteria of the genus Staphylococcus, which has the property of binding to antibodies (immunoglobulins), is widely used (Patent Document 1). However, since Protein A is a protein that specifically binds to the Fc region of antibodies, it cannot be applied to the purification of antibodies without an Fc region, such as single-chain Fv (scFv), Fab, F(ab’)2, IgA, and bispecific T cell engager (BiTE) antibodies. On the other hand, Protein L (hereinafter also referred to as "FpL") derived from bacteria of the genus Finegoldia is a protein that binds to the κ light chain of immunoglobulins. By using FpL as the ligand protein, it becomes possible to purify antibodies without an Fc region, which could not be purified by Protein A described above (Patent Document 2).

[0005] Methods for preserving Fcγ receptors, which are antibody-binding proteins, have been disclosed so far (Cited Documents 3 and 4), but the preservation method of FpL has not been sufficiently studied.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] Provided is a preservation solution that improves the stability of a polypeptide containing at least an immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, it has been found that by including saccharides in the preservation solution, the stability of a polypeptide containing at least an immunoglobulin-binding domain of FpL can be improved, leading to the completion of the present invention.

[0009] That is, the present invention includes the following aspects. [1] A preservation solution for a polypeptide containing at least an immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia, The preservation solution containing 0.03 mol / L or more and 3.0 mol / L or less of saccharides. [2] The preservation solution according to claim 1, wherein the saccharide is one or more selected from trehalose, glucose, and sucrose. [3] The preservation solution according to claim 2, wherein the saccharide is trehalose. [4] A method for preserving the polypeptide, comprising a step of contacting a polypeptide containing at least the immunoglobulin-binding domain of FpL with the preservation solution according to any one of claims 1 to 3. [5] A step of applying a solution containing a polypeptide containing at least the immunoglobulin-binding domain of FpL to a chromatographic carrier-packed column to adsorb the polypeptide to the carrier; A step of applying an elution buffer to the column to elute the polypeptide adsorbed to the carrier; A step of collecting a fraction containing the eluted polypeptide; A step of storing the collected fraction, and A method for producing a polypeptide containing at least the immunoglobulin-binding domain of FpL, wherein the step of storing is a step of storing with the preservation solution according to any one of claims 1 to 3. [6] The preservation solution according to any one of claims 1 to 3, wherein the polypeptide containing at least the immunoglobulin-binding domain of FpL is a polypeptide according to any one of the following (a) to (c); (a) A polypeptide containing at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) A polypeptide containing at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions in the amino acid residue, provided that specific mutations are maintained, and having antibody-binding activity; (c) A polypeptide containing at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, having an amino acid sequence having 70% or more identity to the amino acid sequence consisting of the amino acid residue, provided that specific mutations are maintained, and having antibody-binding activity. [Effect of the Invention]

[0010] The present invention is characterized in that a saccharide is included in a preservation solution of a polypeptide containing at least an immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia. According to the present invention, the stability of FpL in the preservation solution can be improved.

Mode for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail.

[0012] The present invention is characterized in that a saccharide having a concentration of 0.03 mol / L or more and 3.0 mol / L or less is included in a preservation solution of a polypeptide containing at least an immunoglobulin-binding domain of FpL.

[0013] Examples of the saccharide include monosaccharides such as glucose, fructose, and galactose, and disaccharides such as maltose, sucrose, trehalose, cellobiose, lactose, and isomaltose. In food labeling standards, saccharides are defined as monosaccharides or disaccharides and not sugar alcohols. It is preferable in terms of further improving the stability of FpL to include one or more saccharides selected from trehalose, glucose, and sucrose in the preservation solution.

[0014] The concentration of the saccharide that is preferably included in the preservation solution is 0.03 mol / L or more and 3.0 mol / L or less, more preferably 0.05 mol / L or more and 2.0 mol / L, more preferably 0.05 mol / L or more and 1.6 mol / L or less, and still more preferably 0.05 mol / L or more and 1.0 mol / L or less.

[0015] Examples of the polypeptide containing the immunoglobulin-binding domain of FpL of the present invention include the following (a) to (i). (a) A polypeptide containing at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1 (b) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, provided that in the amino acid residue, there is an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, provided that specific mutations are maintained, and having antibody-binding activity. (c) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, provided that it has at least 70% identity to the amino acid sequence consisting of the amino acid residue, provided that specific mutations are maintained, and having antibody-binding activity. (d) A polypeptide comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 2. (e) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 2, provided that in the amino acid residue, there is an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, provided that specific mutations are maintained, and having antibody-binding activity. (f) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 2, provided that it has at least 70% identity to the amino acid sequence consisting of the amino acid residue, provided that specific mutations are maintained, and having antibody-binding activity. (g) A polypeptide comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 7. (h) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 7, provided that in the amino acid residue, there is an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, provided that specific mutations are maintained, and having antibody-binding activity. (i) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 7, provided that it has at least 70% identity to the amino acid sequence consisting of the amino acid residue, provided that specific mutations are maintained, and having antibody-binding activity.

[0016] Specific examples of (b) above include: Proteins that have one or more of the following amino acid substitutions with respect to natural-type FpL_C3 (SEQ ID NO: 1) and have immunoglobulin-binding activity. (1) The amino acid residue corresponding to asparagine at position 50 of SEQ ID NO: 1 is substituted with tyrosine (2) The amino acid residue corresponding to glutamic acid at position 1 of SEQ ID NO: 1 is substituted with glycine or valine (3) The amino acid residue corresponding to proline at position 3 of SEQ ID NO: 1 is substituted with serine (4) The amino acid residue corresponding to glutamic acid at position 4 of SEQ ID NO: 1 is substituted with lysine or glycine (5) The amino acid residue corresponding to glutamic acid at position 5 of SEQ ID NO: 1 is substituted with valine (6) The amino acid residue corresponding to lysine at position 7 of SEQ ID NO: 1 is substituted with alanine (7) The amino acid residue corresponding to glutamic acid at position 8 of SEQ ID NO: 1 is substituted with glycine (8) The amino acid residue corresponding to glutamic acid at position 9 of SEQ ID NO: 1 is substituted with valine (9) The amino acid residue corresponding to isoleucine at position 17 of SEQ ID NO: 1 is substituted with phenylalanine (10) The amino acid residue corresponding to glycine at position 21 of SEQ ID NO: 1 is substituted with arginine (11) The amino acid residue corresponding to glutamic acid at position 27 of SEQ ID NO: 1 is substituted with glycine or arginine (12) The amino acid residue corresponding to lysine at position 29 of SEQ ID NO: 1 is substituted with proline (13) The amino acid residue corresponding to threonine at position 31 of SEQ ID NO: 1 is substituted with leucine (14) The amino acid residue corresponding to threonine at position 36 of SEQ ID NO: 1 is substituted with alanine (15) The amino acid residue corresponding to alanine at position 41 of SEQ ID NO: 1 is substituted with threonine (16) The amino acid residue corresponding to asparagine at position 44 of SEQ ID NO: 1 is substituted with serine or glycine (17) The amino acid residue corresponding to the 49th glutamic acid of SEQ ID NO: 1 is substituted with threonine or isoleucine (18) The amino acid residue corresponding to the 50th asparagine of SEQ ID NO: 1 is substituted with any one of serine, lysine, and aspartic acid (19) The amino acid residue corresponding to the 51st glycine of SEQ ID NO: 1 is substituted with valine (20) The amino acid residue corresponding to the 52nd glutamic acid of SEQ ID NO: 1 is substituted with any one of valine, glycine, and aspartic acid (21) The amino acid residue corresponding to the 53rd tyrosine of SEQ ID NO: 1 is substituted with phenylalanine (22) The amino acid residue corresponding to the 54th threonine of SEQ ID NO: 1 is substituted with isoleucine or methionine (23) The amino acid residue corresponding to the 62nd asparagine of SEQ ID NO: 1 is substituted with any one of histidine, arginine, leucine, methionine, and tryptophan (24) The amino acid residue corresponding to the 65th asparagine of SEQ ID NO: 1 is substituted with tyrosine (25) The amino acid residue corresponding to the 69th alanine of SEQ ID NO: 1 is substituted with threonine (26) The amino acid residue corresponding to the 2nd threonine of SEQ ID NO: 1 is substituted with serine or proline (27) The amino acid residue corresponding to the 3rd proline of SEQ ID NO: 1 is substituted with arginine (28) The amino acid residue corresponding to the 5th glutamic acid of SEQ ID NO: 1 is substituted with lysine or arginine (29) The amino acid residue corresponding to the 27th glutamic acid of SEQ ID NO: 1 is substituted with any one of valine, lysine, and isoleucine (30) The amino acid residue corresponding to the 32nd phenylalanine of SEQ ID NO: 1 is substituted with leucine (31) The amino acid residue corresponding to the 38th lysine of SEQ ID NO: 1 is substituted with alanine (32) The amino acid residue corresponding to the 44th asparagine of SEQ ID NO: 1 is substituted with isoleucine, (33) The amino acid residue corresponding to the 47th alanine of SEQ ID NO: 1 is substituted with threonine or arginine (34) The amino acid residue corresponding to the 52nd glutamic acid of SEQ ID NO: 1 is substituted with asparagine (35) The amino acid residue corresponding to the 2nd threonine of SEQ ID NO: 1 is substituted with alanine (36) The amino acid residue corresponding to the 5th glutamic acid of SEQ ID NO: 1 is substituted with aspartic acid (37) The amino acid residue corresponding to the 6th proline of SEQ ID NO: 1 is substituted with leucine or threonine (38) The amino acid residue corresponding to the 7th lysine of SEQ ID NO: 1 is substituted with proline (39) The amino acid residue corresponding to the 14th valine of SEQ ID NO: 1 is substituted with alanine (40) The amino acid residue corresponding to the 23rd isoleucine of SEQ ID NO: 1 is substituted with arginine or valine (41) The amino acid residue corresponding to the 29th lysine of SEQ ID NO: 1 is substituted with phenylalanine (42) The amino acid residue corresponding to the 31st threonine of SEQ ID NO: 1 is substituted with methionine (43) The amino acid residue corresponding to the 33rd glutamic acid of SEQ ID NO: 1 is substituted with any one of aspartic acid, glycine and valine (44) The amino acid residue corresponding to the 35th alanine of SEQ ID NO: 1 is substituted with threonine (45) The amino acid residue corresponding to the 36th threonine of SEQ ID NO: 1 is substituted with serine (46) The amino acid residue corresponding to the 37th alanine of SEQ ID NO: 1 is substituted with threonine (47) The amino acid residue corresponding to the 41st alanine of SEQ ID NO: 1 is substituted with isoleucine or tyrosine (48) The amino acid residue corresponding to the 44th asparagine of SEQ ID NO: 1 is substituted with histidine or arginine (49) The amino acid residue corresponding to the 52nd glutamic acid of SEQ ID NO: 1 is substituted with leucine or tyrosine (50) The amino acid residue corresponding to the 60th glycine of SEQ ID NO: 1 is substituted with arginine (51) The amino acid residue corresponding to the 64th isoleucine of SEQ ID NO: 1 is substituted with leucine (52) The amino acid residue corresponding to the 66th isoleucine of SEQ ID NO: 1 is substituted with valine (53) The amino acid residue corresponding to the 69th alanine of SEQ ID NO: 1 is substituted with leucine or valine. (54) The amino acid residue corresponding to the 7th lysine of SEQ ID NO: 1 is substituted with glutamine (55) The amino acid residue corresponding to the 13th lysine of SEQ ID NO: 1 is substituted with valine (56) The amino acid residue corresponding to the 29th lysine of SEQ ID NO: 1 is substituted with valine or isoleucine (57) The amino acid residue corresponding to the 6th proline of SEQ ID NO: 1 is substituted with alanine (58) The amino acid residue corresponding to the 8th glutamic acid of SEQ ID NO: 1 is substituted with arginine (59) The amino acid residue corresponding to the 15th asparagine of SEQ ID NO: 1 is substituted with valine (60) The amino acid residue corresponding to the 23rd isoleucine of SEQ ID NO: 1 is substituted with phenylalanine or leucine (61) The amino acid residue corresponding to the 34th glutamic acid of SEQ ID NO: 1 is substituted with any one of aspartic acid, phenylalanine, leucine, and valine (62) The amino acid residue corresponding to the 38th lysine of SEQ ID NO: 1 is substituted with leucine (63) The amino acid residue corresponding to the 50th asparagine of SEQ ID NO: 1 is substituted with methionine (64) The amino acid residue corresponding to the 53rd tyrosine of SEQ ID NO: 1 is substituted with serine. (65) The amino acid residue corresponding to the 49th glutamic acid of SEQ ID NO: 1 is substituted with aspartic acid. (66) The amino acid residue corresponding to the 6th proline of SEQ ID NO: 1 is substituted with serine (67) The amino acid residue corresponding to the 44th asparagine of SEQ ID NO: 1 is substituted with aspartic acid (68) The amino acid residue corresponding to the 49th glutamic acid of SEQ ID NO: 1 is substituted with valine. Substitute tyrosine for the amino acid residue corresponding to asparagine at position 62 of SEQ ID NO: 1. Substitute aspartic acid for the amino acid residue corresponding to asparagine at position 23 of SEQ ID NO: 1. Substitute arginine for the amino acid residue corresponding to lysine at position 13 of SEQ ID NO: 1. Substitute arginine for the amino acid residue corresponding to lysine at position 38 of SEQ ID NO: 1. Substitute arginine for the amino acid residue corresponding to lysine at position 48 of SEQ ID NO: 1. Substitute arginine for the amino acid residue corresponding to lysine at position 67 of SEQ ID NO: 1. Substitute arginine for the amino acid residue corresponding to lysine at position 22 of SEQ ID NO: 1. Substitute glutamic acid for the amino acid residue corresponding to lysine at position 38 of SEQ ID NO: 1.

[0017] Also included are polypeptides (SEQ ID NO: 2) in which amino acid substitutions of Glu4Gly, Pro6Ser, Lys7Ala, Lys13Arg, Lys22Arg, Lys29Ile, Lys38Glu, Lys48Arg, Glu49Asp, Asn50Tyr, Tyr53Phe, Asn62Tyr, Lys67Arg and Ala69Val are introduced into native FpL_C3 (SEQ ID NO: 1), and polypeptides (SEQ ID NO: 7) in which amino acid substitutions of Glu4Gly, Pro6Ser, Lys7Ala, Lys13Arg, Lys22Arg, Ile23Arg, Lys29Ile, Lys38Glu, Asp44Arg, Lys48Arg, Glu49Asp, Asn50Tyr, Tyr53Phe, Asn62Tyr, Lys67Arg and Ala69Val are introduced.

[0018] The "one or several" described in (b), (e) and (h) above may mean, for example, 1 to 30, 1 to 20, or 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.

[0019] The identity of the amino acid sequences described in (c), (f) and (i) may be 70% or more, and may have an identity of more than that, for example, 80% or more, 85% or more, 90% or more, or 95% or more.

[0020] As used herein, the "identity" of amino acid sequences means that two amino acid sequences to be compared are aligned so that as many amino acid residues as possible match, and the percentage is obtained by dividing the number of matching amino acid residues by the total number of amino acid residues. When performing the above alignment, a gap may be appropriately inserted into one or both of the two sequences to be compared as necessary. The method for aligning such sequences is not particularly limited, and for example, it can be performed using well-known sequence comparison programs such as BLAST, FASTA, and CLUSTAL W. When a gap is inserted, the total number of amino acid residues is the number of residues counted with one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the sequence identity (%) is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues of the longer sequence.

[0021] The polypeptide having at least the immunoglobulin-binding domain of the present invention may further have a conservative substitution in which substitution occurs between amino acids having similar physical properties and chemical properties of both amino acids or either of them. Conservative substitutions are generally known to those skilled in the art to maintain the function of the protein between those in which substitution has occurred and those in which substitution has not occurred, not limited to Fc-binding proteins. Examples of conservative substitutions include substitutions that occur between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International Co., Ltd., 9, 2005).

[0022] The polypeptide of the present invention may contain only one immunoglobulin binding domain, or may contain a plurality of immunoglobulin binding domains. The protein of the present invention may contain, for example, two or more, three or more, four or more, or five or more immunoglobulin binding domains, and may contain ten or less, seven or less, five or less, four or less, three or less, or two or less immunoglobulin binding domains, and may contain any non-contradictory combination of these numbers. When the polypeptide of the present invention contains a plurality of immunoglobulin binding domains, the amino acid sequences of these plurality of immunoglobulin binding domains may be the same or different. These plurality of modified amino acid sequences may be in a directly linked (directly connected) form, or may be in a form linked via an appropriate linker (for example, an oligopeptide consisting of 5 to 25 amino acid residues).

[0023] The polypeptide of the present invention may contain, for example, an oligopeptide useful for specifically detecting or separating a target substance at its N-terminal side or C-terminal side. Examples of such oligopeptides include polyhistidine and polyarginine. The protein of the present invention may also contain, for example, an oligopeptide useful for immobilizing the protein of the present invention on a solid phase such as a support for chromatography at its N-terminal side or C-terminal side. Examples of such oligopeptides include oligopeptides containing lysine residues or cysteine residues.

[0024] The polypeptide of the present invention may contain, for example, a part of another immunoglobulin binding domain in addition to the selected immunoglobulin binding domain. For example, when the polypeptide of the present invention contains a modified amino acid sequence of domain C3 of FpL, the protein of the present invention may further contain a part of the N-terminal region (domain C1, domain C2) of domain C3 of FpL, or may contain a part of the C-terminal region (domain C4) of domain C3 of FpL.

[0025] The method for producing the polypeptide of the present invention includes a purification step using a chromatographic carrier-packed column. As an example of a solution containing a polypeptide containing at least the immunoglobulin-binding domain of FpL to be applied to the column, a solution roughly purified from a culture broth of a host (transformant) transformed with an expression plasmid containing a polynucleotide encoding the polypeptide can be mentioned.

[0026] The host is not particularly limited as long as it can express the protein of the present invention by being transformed with the polynucleotide of the present invention. Examples of the host include animal cells, insect cells, and microorganisms. Among these, 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. Further, examples of yeast include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Pichia such as Pichia Pastoris, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe; and examples of bacteria include bacteria of the genus Escherichia such as Escherichia coli. Examples of Escherichia coli include the JM109 strain and the BL21(DE3) strain. Note that using yeast or Escherichia coli as the host is preferable in terms of productivity, and using Escherichia coli as the host is even more preferable.

[0027] To obtain a solution containing a polypeptide containing at least the immunoglobulin-binding domain of FpL, which is applied to a carrier-packed column for chromatography, from the culture solution of the transformant, it may be appropriately selected depending on the form of expression. For example, when the expressed polypeptide leaks from the periplasm of the host cell into the culture supernatant, the cells may be separated by a centrifugation operation, and the protein of the present invention may be recovered from the obtained culture supernatant. Further, when the protein of the present invention accumulates intracellularly (including the periplasm), after the cells are recovered by a centrifugation operation, an enzyme treatment agent, a surfactant, or the like is added to disrupt the cells, and the protein of the present invention may be recovered from the disrupted product.

[0028] The recovery of the polypeptide of the present invention from the above-described culture supernatant or cell lysate is not particularly limited as long as the carrier-packed column for chromatography is a column packed with a carrier for chromatography that is usually used for protein purification by those skilled in the art. Examples of the carrier include a carrier for gel filtration chromatography, a carrier for cation exchange chromatography, a carrier for anion chromatography, a carrier for hydrophobic chromatography, and a carrier for affinity chromatography.

Example

[0029] Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited to these examples.

[0030] Example 1 It was examined whether the polypeptide was stabilized by adding a saccharide to a solution containing a polypeptide containing at least the immunoglobulin-binding domain of Protein L (FpL) derived from a bacterium of the genus Finegoldia.

[0031] (1) Escherichia coli BL21(DE3) strain was transformed with an expression vector containing a polynucleotide encoding (FpL_C3KX 7e)4-IT-6H3K (SEQ ID NO: 5), which is a polypeptide containing at least the immunoglobulin-binding domain of FpL, and an inducible promoter, to prepare a genetically recombinant Escherichia coli capable of expressing the protein.

[0032] In addition, (FpL_C3KX 7e)4-IT-6H3K (SEQ ID NO: 5) is a polypeptide in which four FpL_C3KX 7e (SEQ ID NO: 2), which is an amino acid substitution variant of the domain C3 of FpL (amino acid residues from position 394 to position 463 of GenBank No. AAA67503, SEQ ID NO: 1, hereinafter also referred to as "FpL_C3"), are linked in series, and an oligopeptide (SEQ ID NO: 3), a histidine tag (SEQ ID NO: 4), and three lysine residues for improving the immobilization rate on an insoluble carrier are linked in this order to the C-terminus thereof. FpL_C3KX 7e (SEQ ID NO: 2) is an immunoglobulin-binding protein in which amino acid substitutions of E4G, P6S, K7A, K13R, K22R, K29I, K38E, K48R, E49D, N50Y, Y53F, N62Y, K67R, and A69V are introduced with respect to FpL_C3 (SEQ ID NO: 1) (Japanese Patent Laid-Open No. 2023-064059).

[0033] (2) The Escherichia coli was inoculated into 20 mL of 2×YT liquid medium (tryptone 1.6% (w / v), yeast extract 1% (w / v), sodium chloride 0.5% (w / v)) containing 50 μg / mL of kanamycin and pre-cultured (30°C, 16 hours).

[0034] (3) 10 mL of the pre-cultured culture solution was inoculated into 1 L of 2×YT liquid medium containing 50 μg / mL of kanamycin and cultured at 37°C for 2 to 3 hours. Then, 200 μL of 0.5 M IPTG (Isopropyl-β-D-thiogalactopyranoside) was added, and further cultured with shaking overnight at 20°C.

[0035] (4) The cultured culture solution was centrifuged to recover the cultured cells (wet weight: 8 g). 2 g of the cultured cells in terms of wet weight were lysed using BugBuster Protein Extraction Reagent (manufactured by Merck Millipore), and the supernatant obtained by centrifugation was passed through a filter to clarify it.

[0036] (5) The supernatant clarified in (2-3) was added to a column packed with Ni Sepharose 6 Fast Flow (manufactured by Cytiva) equilibrated in advance with 0.02 M Tris-HCl buffer (pH 7.5) containing 0.5 M sodium chloride and 0.02 M imidazole (hereinafter also simply referred to as "equilibration solution"). After washing with 10 times the volume of the equilibration solution of the carrier packed in the column, 0.02 M Tris-HCl buffer (pH 7.5) containing 0.5 M sodium chloride and 0.5 M imidazole was passed through, and the fraction corresponding to the immunoglobulin-binding protein was recovered.

[0037] (6) (FpL_C3KX 7e)4-IT-6H3K (SEQ ID NO: 5), a polypeptide containing at least the immunoglobulin-binding domain of FpL, was subjected to solvent replacement with a buffer solution of pH 6.8 containing 15 mmol / L sodium phosphate and 200 mmol / L sodium chloride (hereinafter also referred to as "buffer solution A") using Amicon Ultra (manufactured by Merck Millipore). After that, the concentration of (FpL_C3KX 7e)4-IT-6H3K was adjusted to 25 g / L.

[0038] (7) The solution containing (FpL_C3KX 7e)4-IT-6H3K prepared in (6) was diluted with buffer solution A containing saccharides so that the concentration of the polypeptide became 1 g / L (hereinafter also simply referred to as "diluted sample"). The saccharides used and their concentrations are as follows. Saccharides: Any of glucose, sucrose, and trehalose Concentration (final concentration contained in the diluted sample): Any of 0.060 mol / L, 0.12 mol / L, 0.25 mol / L, 0.50 mol / L, 1.0 mol / L, 1.2 mol / L, and 1.5 mol / L (8) The diluted sample prepared in (7) was measured for the thermal denaturation midpoint temperature (Tm), which is an index of the thermal stability of the protein, by fluorescence spectrum measurement using UNcle (manufactured by Unchained Labs).

[0039] Comparative Example 1 In Example 1(7), the Tm of the diluted sample was measured in the same manner as in Example 1, except that it was diluted with buffer A containing arginine sulfate or glycerol instead of buffer A containing saccharides. The concentration of the arginine sulfate or glycerol was the same as in Example 1(7).

[0040] Comparative Example 2 In Example 1(7), the Tm of the diluted sample was measured in the same manner as in Example 1, except that it was diluted with buffer A not containing additives such as saccharides instead of buffer A containing saccharides.

[0041] The results of Example 1 and Comparative Examples 1 and 2 are shown together in Table 1. When saccharides (glucose, sucrose, or trehalose) were added (Example 1), the Tm increased compared to when not added (Comparative Example 2, 82.8 °C) at any saccharide and added concentration, and the Tm increased depending on the added concentration. From the above results, it can be seen that when saccharides are added to the storage solution of the polypeptide containing at least the immunoglobulin-binding domain of FpL at a final concentration of 0.03 mol / L or more and 3.0 mol / L or less, the stability of the polypeptide in the solution is improved.

[0042] On the other hand, when arginine sulfate or glycerol was added (Comparative Example 1), the Tm decreased compared to when not added (Comparative Example 2, 82.8 °C) at any added concentration. From the above results, it can be seen that when an amino acid or sugar alcohol is added to a solution containing a polypeptide containing at least the immunoglobulin-binding domain of FpL, the stability of the polypeptide decreases.

[0043]

Table 1

[0044] Example 2 A stabilization test was conducted using a polypeptide different from the polypeptide used in Example 1.

[0045] (1) A polypeptide containing at least the immunoglobulin-binding domain of FpL, a polynucleotide encoding (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 6), and an inducible promoter were used to transform Escherichia coli BL21(DE3) strain, and a recombinant Escherichia coli capable of expressing the protein was prepared.

[0046] Note that (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 6) is a polypeptide obtained by adding an immobilization tag peptide (IT) consisting of the amino acid sequence set forth in SEQ ID NO: 3 and three lysine residues (3K) to the C-terminal side of a pentamer ((FpL_C3KX 9a)5) in which five polypeptides FpL_C3KX 9a (SEQ ID NO: 7) (WO2023 / 074642), in which the following amino acid substitutions (i) to (xvi) occurred with respect to the immunoglobulin-binding domain C3 of FpL (amino acid residues 394 to 463 of GenBank No. AAA67503, SEQ ID NO: 1), are directly linked. (Hereinafter, it is also referred to as (FpL_C3KX 9a)5-IT-3K). (i) Glutamic acid at the 4th position of SEQ ID NO: 1 is substituted with glycine (ii) Proline at the 6th position of SEQ ID NO: 1 is substituted with serine (iii) Lysine at the 7th position of SEQ ID NO: 1 is substituted with alanine (iv) Lysine at the 13th position of SEQ ID NO: 1 is substituted with arginine (v) Lysine at the 22nd position of SEQ ID NO: 1 is substituted with arginine (vi) Isoleucine at the 23rd position of SEQ ID NO: 1 is substituted with arginine (vii) Lysine at the 29th position of SEQ ID NO: 1 is substituted with isoleucine (viii) Lysine at the 38th position of SEQ ID NO: 1 is substituted with glutamic acid (ix) Asparagine at the 44th position of SEQ ID NO: 1 is substituted with arginine (x) Lysine at the 48th position of SEQ ID NO: 1 is substituted with arginine (xi) Glutamic acid at the 49th position of SEQ ID NO: 1 is substituted with aspartic acid (xii) Asparagine at the 50th position of SEQ ID NO: 1 is substituted with tyrosine (xiii) The 53rd tyrosine of SEQ ID NO: 1 is replaced with phenylalanine (xiv) The 62nd asparagine of SEQ ID NO: 1 is replaced with tyrosine (xv) The 67th lysine of SEQ ID NO: 1 is replaced with arginine (xvi) The 69th alanine of SEQ ID NO: 1 is replaced with valine (2) 20 mL of 2×YT liquid medium (tryptone 1.6% (w / v), yeast extract 1% (w / v), sodium chloride 0.5% (w / v)) containing 50 μg / mL of kanamycin was inoculated with the E. coli and pre-cultured (30 °C, 16 h).

[0047] (3) 10 mL of the pre-cultured culture broth was inoculated into 1 L of 2×YT liquid medium containing 50 μg / mL of kanamycin and cultured at 37 °C for 2 to 3 h, then 200 μL of 0.5 M IPTG (Isopropyl-β-D-thiogalactopyranoside) was added, and further cultured with shaking overnight at 20 °C.

[0048] (4) The cultured culture broth was centrifuged to recover the cultured cells (8 g in wet weight). 2 g of the cultured cells in wet weight were lysed using BugBuster Protein Extraction Reagent (manufactured by Merck Millipore), and the supernatant obtained by centrifugation was passed through a filter to clarify it.

[0049] (5) The supernatant clarified in (2 - 3) was added to a column packed with IgG Sepharose 6 Fast Flow (manufactured by Cytiva) pre-equilibrated with 0.02 M Tris-HCl buffer (pH 7.5) containing 0.15 M sodium chloride (hereinafter also simply referred to as "equilibration solution"). After washing with 10 times the volume of the equilibration solution of the carrier packed in the column, an acid solution (pH 2.2) containing 0.1 M glycine hydrochloride was passed through, and the fraction corresponding to the immunoglobulin-binding protein was recovered. (6) The polypeptide (FpL_C3KX 9a)5-IT-3K that contains at least the immunoglobulin-binding domain of FpL was subjected to solvent replacement using Amicon Ultra (manufactured by Merck Millipore) into a buffer solution of pH 6.8 containing 15 mmol / L sodium phosphate and 200 mmol / L sodium chloride (hereinafter also referred to as "buffer solution A"), and then the concentration of (FpL_C3KX 9a)5-IT-3K was adjusted to 25 g / L.

[0050] (7) The solution containing (FpL_C3KX 9a)5-IT-3K prepared in (6) was diluted with buffer solution A containing saccharides so that its concentration became 1 g / L (hereinafter also simply referred to as "diluted sample"). The saccharides used and their concentrations are as follows. Saccharides: Any one of glucose, sucrose, and trehalose Concentration (final concentration contained in the diluted sample): Any one of 0.25 mol / L, 0.50 mol / L, 1.0 mol / L, 2.0 mol / L, and 4.0 mol / L (8) The diluted sample prepared in (7) was measured for the thermal denaturation midpoint temperature (Tm), which is an index of the thermal stability of the protein, by fluorescence spectrum measurement using UNcle (Unchained Labs).

[0051] Comparative Example 3 In Example 2 (7), except that it was diluted with buffer solution A containing no additives such as saccharides instead of buffer solution A containing saccharides, the Tm of the diluted sample was measured in the same manner as in Example 2.

[0052] The results of Example 2 and Comparative Example 3 are shown together in Table 2. When saccharides (glucose, sucrose, or trehalose) were added (Example 2), the Tm increased in any of the saccharides as compared with the case without addition (82.3 °C). Also, in the concentration range of saccharides from 0.25 mol / L to 2.0 mol / L, the Tm increased depending on the concentration.

[0053] From the above results, it can be seen that when saccharides are added to the storage solution of the polypeptide containing at least the immunoglobulin-binding domain at a final concentration of 0.03 mol / L or more and 3.0 mol / L or less, the stability of the polypeptide in the solution is improved. On the other hand, when 4.0 mol / L of glucose is added, it is shown that the Tm decreases compared to the case where no saccharides are added.

[0054] The polypeptide (FpL_C3KX 7e) 4-IT-6H3K (SEQ ID NO: 5) used in Example 1 and the polypeptide (FpL_C3KX 9a) 5-IT-3K (SEQ ID NO: 6) used in Example 2 differ in the mutation position, number of mutations, number of monomers, and additional sequence on the C-terminal side of the immunoglobulin-binding domain, but the Tm increases with the addition of saccharides. From the above, it is shown that the storage stability of the polypeptide containing the immunoglobulin-binding domain is improved by adding saccharides to the storage solution of the polypeptide, regardless of the mutation position, number of mutations, number of monomers, and additional sequence on the C-terminal side of the immunoglobulin-binding domain.

[0055] [Table 2]

Claims

1. A preservation solution for a polypeptide comprising at least an immunoglobulin-binding domain of Protein L (FpL) derived from a bacterium of the genus Finegoldia, The preservation solution containing saccharides at 0.03 mol / L or more and 3.0 mol / L or less.

2. The preservation solution according to Claim 1, wherein the saccharide is one or more selected from trehalose, glucose, and sucrose.

3. The preservation solution according to Claim 2, wherein the saccharide is trehalose.

4. A method for preserving the polypeptide, comprising the step of contacting a polypeptide comprising at least an immunoglobulin-binding domain of FpL with the preservation solution according to any one of Claims 1 to 3.

5. Applying a solution containing a polypeptide comprising at least an immunoglobulin-binding domain of FpL to a carrier-packed column for chromatography and adsorbing the polypeptide to the carrier; Applying an elution buffer to the column and eluting the polypeptide adsorbed to the carrier; Recovering the fraction containing the eluted polypeptide; A method for producing a polypeptide comprising at least an immunoglobulin-binding domain of FpL, comprising the step of storing the recovered fraction, wherein the step of storing is a step of storing with the preservation solution according to any one of Claims 1 to 3.

6. The preservation solution according to any one of Claims 1 to 3, wherein the polypeptide comprising at least an immunoglobulin-binding domain of FpL is a polypeptide according to any one of the following (a) to (c); (a) A polypeptide comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, (b) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, and having one or several substitutions, deletions, insertions, or additions of one or several amino acid residues at one or several positions in the amino acid residue, provided that a specific mutation is maintained, and having antibody-binding activity, (c) A polypeptide having an amino acid sequence comprising at least an amino acid residue consisting of the amino acid sequence set forth in SEQ ID NO: 1, having an identity of 70% or more with respect to the amino acid sequence consisting of the amino acid residue, provided that a specific mutation is maintained, and having antibody-binding activity. ​

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