Immunoglobulin-binding protein production method

Abstract

To provide a method that enables efficient production of a polypeptide containing at least an immunoglobulin-binding domain of Protein L (FpL) originating from Finegoldia bacteria.SOLUTION: A production method comprises the step of culturing a recombinant Escherichia coli containing a polynucleotide encoding a polypeptide including at least an immunoglobulin-binding domain of FpL and expressing the polypeptide, and the step of purifying the polypeptide from a culture of the Escherichia coli, wherein the step includes a purification step using an anion-exchange chromatography and a purification step using a hydrophobic chromatography, wherein the purification step using the hydrophobic chromatography includes a step of equilibrating a hydrophobic chromatography column, a step of adding a sample containing the polypeptide to the column, and a step of recovering the polypeptide, wherein at least one of a solution for equilibrating the column and the sample contains 0.4 mol / L-1.1 mol / L ammonium sulfate.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present invention relates to a method for producing an immunoglobulin-binding protein, and in particular to a method for efficiently producing said protein. [Background technology]

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

[0003] The production of antibody drugs involves a culture and purification process, during which antibody-producing cells are modified and culture conditions are optimized to improve productivity. The purification process employs affinity chromatography for crude purification, followed by intermediate purification, final purification, and virus removal before being formulated.

[0004] The purification process uses an affinity carrier that specifically recognizes antibody molecules. Protein A (hereinafter also referred to as SpA), derived from Staphylococcus bacteria, which has the property of binding to antibodies (immunoglobulins), is often used as the ligand protein used in the carrier (Patent Document 1). However, because SpA specifically binds to the Fc region of antibodies, it cannot be applied to the purification of antibodies that do not have an Fc region, such as single-chain Fv (scFv), Fab, F(ab')2, IgA, and bispecific T cell-triggering (BiTE) antibodies.

[0005] On the other hand, Protein L (hereinafter referred to as FpL) derived from bacteria of the genus Finegoldia is a protein that binds to the κ light chain of immunoglobulin. By using FpL as a ligand protein, it is possible to purify antibodies that do not have an Fc region, which cannot be purified using the aforementioned SpA (Patent Document 2).

[0006] As techniques related to the production of FpL, Patent Documents 3 and 4 disclose a method for purifying Protein L derived from Peptostreptococcus magnus (Finegoldia magna) by performing cation exchange chromatography purification followed by anion exchange chromatography purification. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2010-504754 [Patent Document 2] WO2017 / 191748 issue [Patent Document 3] WO2017 / 069158 issue [Patent Document 4] WO2016 / 121703 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, Patent Documents 3 and 4 disclose methods for purifying Protein L by cation exchange chromatography followed by anion exchange chromatography. However, the present inventors have found that, depending on the form of Protein L, purification by cation exchange chromatography may be difficult.

[0009] Furthermore, the methods disclosed in Patent Documents 3 and 4 require that anion exchange chromatography be performed by varying the pH of the eluate after cation exchange chromatography purification and then applying the eluate with the pH varied to an anion exchange carrier equilibrated at the pH after the pH variation. Such pH variation may cause the target protein to cross its isoelectric point, which may result in aggregation and / or precipitation of the insoluble protein.

[0010] Furthermore, in this method, it may be necessary to reduce the conductivity of the eluate after purification by cation exchange chromatography before performing anion exchange chromatography. In this case, a dialysis treatment is required to reduce the conductivity of the eluate. However, this requires a large amount of dialysis solution, which increases the number of steps, posing a problem for industrial production of proteins using this method.

[0011] Therefore, an object of the present invention is to provide a method for efficiently producing a polypeptide comprising at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia. Another object of one aspect of the present invention is to provide a method for industrially and efficiently producing a polypeptide comprising at least the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia. Another object of one aspect of the present invention is to provide a method for industrially and efficiently producing a polypeptide comprising at least the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia, without pH fluctuation or dialysis in the step of purifying the polypeptide by chromatography from a culture of recombinant E. coli obtained in the step of culturing a recombinant E. coli containing a polynucleotide encoding a polypeptide comprising at least the immunoglobulin-binding domain of Protein L derived from bacteria of the genus Finegoldia and expressing the polypeptide. [Means for solving the problem]

[0012] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that when purifying a polypeptide from a recombinant Escherichia coli culture containing a polynucleotide encoding the polypeptide, the polypeptide contains at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia, the polypeptide can be efficiently produced by including a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography.

[0013] That is, the present invention includes the following aspects. [1] A step of culturing a recombinant Escherichia coli containing a polynucleotide encoding a polypeptide containing at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia, and expressing the polypeptide; and purifying the polypeptide from the culture of the recombinant E. coli obtained in the expression step by using chromatography, the step of purifying the polypeptide comprises a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography; The purification step using hydrophobic chromatography includes: equilibrating a hydrophobic chromatography column, applying a sample containing the polypeptide to the column, and recovering the polypeptide; At least one of the solution for equilibrating the column and the sample contains ammonium sulfate at a concentration of 0.4 mol / L or more and 1.1 mol / L or less. method. [2] The method according to [1], wherein at least one of the solution for equilibrating the column and the sample further contains sodium chloride. [3] The production method according to either [1] or [2], wherein the polypeptide comprising at least the immunoglobulin-binding domain of FpL is a polypeptide according to any one of the following (a) to (c): (a) a polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide having an amino acid sequence containing at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having immunoglobulin-binding activity; (c) A polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, having 70% or more identity to the amino acid sequence consisting of said amino acid residues, and having immunoglobulin-binding activity. [Effects of the Invention]

[0014] According to the present invention, a polypeptide comprising at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia can be efficiently produced.

[0015] In one embodiment of the present invention, the purity of the polypeptide can be improved when it is purified from a culture of recombinant E. coli containing a polynucleotide encoding the polypeptide.In another embodiment of the present invention, the yield of the polypeptide can be improved when it is purified from a culture of recombinant E. coli containing a polynucleotide encoding the polypeptide.

[0016] In another embodiment of the present invention, a method for industrially and efficiently producing the polypeptide can be provided. Furthermore, in another embodiment of the present invention, a method for industrially and efficiently producing the polypeptide can be provided, in which the polypeptide is purified by chromatography from a culture of recombinant E. coli obtained in the step of culturing a recombinant E. coli containing a polynucleotide encoding the polypeptide and expressing the polypeptide, without pH fluctuation or dialysis. In the step of purifying the polypeptide by chromatography, the absence of pH fluctuation eliminates the possibility of purification crossing the isoelectric point of Protein L, thereby suppressing loss of the target protein that would occur by crossing the isoelectric point. [Brief explanation of the drawings]

[0017] [Figure 1]This figure shows the results of SDS-PAGE of fractions eluted from a hydrophobic chromatography column or an anion exchange chromatography column. M indicates molecular weight markers; B indicates a sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) obtained in Example 1; H1 indicates a fraction eluted from a column packed with butyl; H2 indicates a fraction eluted from a column packed with phenyl; H3 indicates a fraction eluted from a column packed with PPG; A1 indicates a fraction eluted from a column packed with SuperQ; A2 indicates a fraction eluted from a column packed with GigaCapQ; and A3 indicates a fraction eluted from a column packed with DEAE. The band indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. [Figure 2] This figure shows the results of SDS-PAGE of the flow-through fraction, wash fraction, and elution fraction obtained by purification using a cation exchange chromatography column. M indicates the molecular weight marker, B indicates the sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) obtained in Example 1, C1 indicates the results obtained using an SP column, C2 indicates the results obtained using a CM column, and C3 indicates the results obtained using a GigaCapS column. T indicates the flow-through fraction, W indicates the results obtained using a wash fraction, and E indicates the results obtained using an elution fraction. The band indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. [Figure 3] This figure shows the effect of different salt concentrations in the buffer used for equilibration on the chromatogram during purification using an anion-exchange chromatography column. (a) Results when the sodium chloride concentration in the buffer used for equilibration was 50 mmol / L, (b) when it was 100 mmol / L, (c) when it was 150 mmol / L, and (d) when it was 200 mmol / L. The elution peak indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. [Figure 4]This figure shows the results of SDS-PAGE of the supernatant and precipitate (precipitate suspension) obtained by centrifuging a sample after adding ammonium sulfate. (FpL_C3KX 9a) The results are shown for a sample containing 5-IT-3K and for equilibration solutions with ammonium sulfate concentrations of 0.3 mol / L, 0.6 mol / L, 0.9 mol / L, 1.2 mol / L, and 1.5 mol / L. [Figure 5] This figure shows the effect of different salts added to the sample on the chromatogram during hydrophobic chromatography purification. (a) shows the results when 0.8 mol / L ammonium sulfate was added, (b) when 0.8 mol / L ammonium sulfate and 1 mol / L sodium chloride were added, and (c) when 0.8 mol / L ammonium sulfate and 2 mol / L sodium chloride were added. The elution peak indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. [Figure 6] 1 is a chromatogram showing the results of the first purification step (purification using an anion exchange chromatography column) in Example 7. (a), (b), (c), and (d) in the figure correspond to the conditions (a), (b), (c), and (d) in Example 7. The elution peak indicated by the white arrow is the peak corresponding to the FpL polypeptide. [Figure 7] 1 is a chromatogram showing the results of the second purification step (purification using a hydrophobic chromatography column) in Example 7. (a), (b), (c), and (d) in the figure correspond to the conditions (a), (b), (c), and (d) in Example 7. The elution peak indicated by the white arrow is the peak corresponding to the FpL polypeptide. [Figure 8] 1 shows chromatograms illustrating the results of purification in Example 8. (a) and (b) in the figure correspond to conditions (a) and (b) in Example 8, with the "1st" chromatogram on the left representing the results of the first purification step (purification using an anion exchange chromatography column) and the "2nd" chromatogram on the right representing the results of the second purification step (purification using a hydrophobic chromatography column). The elution peak indicated by the white arrow corresponds to the FpL polypeptide. [Figure 9]1 shows chromatograms illustrating the results of purification in Example 9. (a) and (b) in the figure correspond to conditions (a) and (b) in Example 9, respectively. The "1st" chromatogram on the left is the result of the first purification step (purification using a hydrophobic chromatography column), and the "2nd" chromatogram on the right is the result of the second purification step (purification using an anion exchange chromatography column). The elution peak indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. [Figure 10] This figure shows the results of SDS-PAGE of fractions obtained by purification under condition (b) of Example 9. Lane M shows the results of molecular weight markers, lane B shows the results of the sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) obtained in Example 1, lane H shows the results of the elution fraction from the first purification step (purification using a hydrophobic chromatography column), and lane A shows the results of the elution fraction from the second purification step (purification using an anion exchange chromatography column). [Figure 11] 1 shows chromatograms illustrating the results of purification in Example 10. (a) and (b) in the figure correspond to conditions (a) and (b) in Example 10. The "1st" chromatogram on the left is the result of the first purification step (purification using a hydrophobic chromatography column), and the "2nd" chromatogram on the right is the result of the second purification step (purification using an anion exchange chromatography column). The elution peak indicated by the white arrow corresponds to (FpL_C3KX 7e)4-IT-3K. [Figure 12] 1 shows the results of SDS-PAGE of fractions obtained by purification under condition (b) of Example 10. Lane M shows the results of molecular weight markers, lane S shows the results of a sample obtained in Example 1 containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) which was purified on a hydrophobic chromatography column and then purified on an anion exchange chromatography column, lane B shows the results of a sample obtained in Example 1 containing (FpL_C3KX 7e)4-IT-3K (SEQ ID NO: 5), lane H shows the results of the elution fraction from the first purification step (purification using a hydrophobic chromatography column), and lane A shows the results of the elution fraction from the second purification step (purification using an anion exchange chromatography column). [Figure 13]1 shows chromatograms illustrating the results of purification in Example 11. The upper "1st" chromatogram shows the results of the first purification step (purification using a hydrophobic chromatography column), and the lower "2nd" chromatogram shows the results of the second purification step (purification using an anion exchange chromatography column). (a), (b), and (c) in the figure correspond to conditions (a), (b), and (c) in Example 11, respectively. The elution peak indicated by the white arrow corresponds to (FpL_C3KX 9a)5-IT-3K. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below.

[0019] As used herein, the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia is (i) It may be a polypeptide containing amino acid residues consisting of the sequence of the domain, As long as it has immunoglobulin-binding activity, (ii) It may be a polypeptide comprising amino acid residues consisting of a partial sequence of the domain. Furthermore, as long as it has binding activity to immunoglobulins, (iii) A polypeptide containing amino acid residues consisting of the sequence of the domain or a partial sequence thereof, wherein the amino acid residues have one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions; (iv) It may be a polypeptide comprising at least amino acid residues of a sequence having 70% or more identity with the sequence of the domain or a partial sequence thereof.

[0020] Hereinafter, the polypeptides described in (iii) and (iv) above will also be collectively referred to as variants.

[0021] In the immunoglobulin-binding domain of FpL, the Finegoldia genus bacterium from which FpL is derived includes Finegoldia magna. The immunoglobulin-binding domain of Protein L derived from Finegoldia magna is as follows: Domain B1 (amino acid residues 104 to 173 of GenBank No. AAA25612), Domain B2 (amino acid residues 176 to 245 of GenBank No. AAA25612), Domain B3 (amino acid residues 248 to 317 of GenBank No. AAA25612), Domain B4 (amino acid residues 320 to 389 of GenBank No. AAA25612), Domain B5 (amino acid residues 393 to 462 of GenBank No. AAA25612), Domain C1 (amino acid residues 249 to 317 of GenBank No. AAA67503), Domain C2 (amino acid residues 320 to 389 of GenBank No. AAA67503), Domain C3 (amino acid residues 394 to 463 of GenBank No. AAA67503: SEQ ID NO: 1), and An example is domain C4 (amino acid residues 468 to 537 of GenBank No. AAA67503), but any domain may be selected.

[0022] The immunoglobulin-binding domain of FpL may contain, for example, a portion of another domain in addition to the selected domain. For example, when the amino acid sequence of immunoglobulin-binding domain C3 of Protein L derived from Finegoldia magna is selected as the immunoglobulin-binding domain of FpL, the immunoglobulin-binding domain of FpL may further contain a portion of the N-terminal region of said domain (domain C1, domain C2), or a portion of the C-terminal region of said domain (domain C4).

[0023] Regarding (ii) above, the immunoglobulin-binding domain of FpL ((i) above) is composed of four β-sheets, one α-helix, a loop connecting them, and an N-terminal loop region. However, amino acid residues in regions unrelated to antibody binding, such as the N-terminal loop region, may be deleted. As a specific example, when the immunoglobulin-binding domain of FpL ((i) above) is domain C3 (SEQ ID NO: 1), it is known that antibody-binding ability is maintained even when the amino acid residues from the first glutamic acid (E) to the ninth glutamic acid (E), which correspond to the N-terminal loop region, are deleted (Housden NG et al., Biochemical Society Transactions, 31, 716-718, 2003). In other words, the partial amino acid sequence in (ii) above is sufficient as long as it contains at least the amino acid sequence of the antibody-binding site. In other words, the partial amino acid sequence in (ii) above is a partial sequence containing the amino acid sequence of the antibody-binding site.

[0024] The term "one or several" in (iii) above varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically means, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.

[0025] The "identity" of amino acid sequences in (iv) above is calculated by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible are identical, and dividing the number of identical amino acid residues by the total number of amino acid residues, expressed as a percentage. During the alignment, gaps may be inserted as needed into one or both of the two sequences to be compared. The method for aligning such sequences is not particularly limited, and can be performed using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, and CLUSTALW. When gaps are inserted, the total number of amino acid residues is calculated by counting each gap as one amino acid residue. If the total number of amino acid residues thus counted differs between the two sequences to be compared, the identity is calculated by dividing the number of identical amino acid residues by the total number of amino acid residues in the longer sequence. The "identity" of amino acid sequences in (iv) above may be 70% or more, but may also be higher, for example, 80% or more, 90% or more, or 95% or more.

[0026] Specific examples of the immunoglobulin-binding domain of FpL include the variants disclosed in WO2023 / 074642 and proteins that contain amino acid residues consisting of the sequence set forth in SEQ ID NO: 1 (immunoglobulin-binding domain C3 of Protein L derived from Finegoldia magna), provided that the amino acid residues have at least one amino acid substitution selected from the following (1) to (4), and that have immunoglobulin-binding activity (WO2024 / 225177). (1) The amino acid residue corresponding to lysine 22 of SEQ ID NO: 1 is substituted with glutamic acid (2) The amino acid residue corresponding to lysine 38 of SEQ ID NO: 1 is substituted with proline or aspartic acid (3) The amino acid residue corresponding to tyrosine at position 42 of SEQ ID NO: 1 is substituted with any one of histidine, tryptophan, and phenylalanine. (4) The amino acid residue corresponding to asparagine 44 of SEQ ID NO: 1 is substituted with proline The immunoglobulin-binding domain of FpL may specifically be a protein selected from any of the following (a) to (f): (a) a polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide having an amino acid sequence containing at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having immunoglobulin-binding activity; (c) A polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, having 70% or more identity to the amino acid sequence consisting of said amino acid residues, and having immunoglobulin-binding activity. (d) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 3; (e) the amino acid sequence shown in SEQ ID NO: 2 or 3, wherein the following amino acid substitutions are present: <1> ~ <16> a protein having an amino acid sequence which further includes any one or more of substitution, deletion, insertion and addition of one or several amino acid residues at one or several positions in addition to the above, and which has immunoglobulin-binding activity; <1> The amino acid residue corresponding to lysine 22 in SEQ ID NO: 1 is substituted with arginine <2> The amino acid residue corresponding to lysine at position 38 of SEQ ID NO: 1 is substituted with glutamic acid <3> The amino acid residue corresponding to asparagine at position 44 of SEQ ID NO: 1 is substituted with arginine <4> The amino acid residue corresponding to the fourth glutamic acid in SEQ ID NO: 1 is substituted with glycine <5> The amino acid residue corresponding to the sixth proline in SEQ ID NO: 1 is substituted with serine <6> The amino acid residue corresponding to the 7th lysine in SEQ ID NO: 1 is substituted with alanine <7> The amino acid residue corresponding to the 13th lysine in SEQ ID NO: 1 is substituted with arginine <8> The amino acid residue corresponding to the 23rd isoleucine in SEQ ID NO: 1 is substituted with arginine <9> The amino acid residue corresponding to lysine at position 29 of SEQ ID NO: 1 is substituted with isoleucine <10> The amino acid residue corresponding to lysine at position 48 of SEQ ID NO: 1 is substituted with arginine <11> The amino acid residue corresponding to glutamic acid at position 49 of SEQ ID NO: 1 is substituted with aspartic acid <12> The amino acid residue corresponding to asparagine at position 50 of SEQ ID NO: 1 is substituted with tyrosine <13> The amino acid residue corresponding to tyrosine at position 53 of SEQ ID NO: 1 is substituted with phenylalanine <14> The amino acid residue corresponding to asparagine at position 62 of SEQ ID NO: 1 is substituted with tyrosine <15> The amino acid residue corresponding to lysine 67 of SEQ ID NO: 1 is substituted with arginine <16> The amino acid residue corresponding to alanine at position 69 of SEQ ID NO: 1 is substituted with valine (f) an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 2 or 3, provided that the above amino acid substitutions are not included. <1> ~ <16> and having immunoglobulin-binding activity.

[0027] The "one or several" in (b) and (e) will vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but may specifically be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0028] Furthermore, the "identity" in (c) and (f) may be, for example, an amino acid sequence that has 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0029] As used herein, "the Xth amino acid of SEQ ID NO: 1" refers to the amino acid located at position X counting from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1. "The amino acid residue corresponding to the Xth amino acid of SEQ ID NO: 1" in a specific amino acid sequence refers to an amino acid residue in the specific amino acid sequence that is arranged at the same position as the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 1 when the specific amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1. Furthermore, in the amino acid sequence of SEQ ID NO: 1, "an amino acid residue corresponding to amino acid X of SEQ ID NO: 1" refers to the amino acid X itself in the amino acid sequence of SEQ ID NO: 1. In other words, the positions of the above-exemplified amino acid substitutions (i.e., amino acid substitutions at the specific positions and, optionally, other amino acid substitutions) do not necessarily indicate absolute positions in the protein, but indicate relative positions based on the amino acid sequence of SEQ ID NO: 1. That is, for example, when a variant of the immunoglobulin-binding domain of FpL contains an insertion, deletion, or addition of an amino acid residue N-terminal to the positions of the above-exemplified amino acid substitutions, the absolute positions of the amino acid substitutions may vary accordingly.

[0030] A polypeptide comprising an FpL immunoglobulin-binding domain may contain only one immunoglobulin-binding domain or multiple immunoglobulin-binding domains. The polypeptide may contain, for example, two or more, three or more, four or more, or five or more immunoglobulin-binding domains, or up to ten, seven or less, five or less, four or less, three or less, or two or less immunoglobulin-binding domains, or any compatible combination thereof. When the polypeptide comprises multiple immunoglobulin-binding domains, the amino acid sequences of the multiple immunoglobulin-binding domains may or may not be identical. The multiple modified amino acid sequences may be directly linked (directly linked) or linked via a suitable linker (e.g., an oligopeptide consisting of 5 to 25 amino acid residues). When a polypeptide comprising at least an FpL immunoglobulin-binding domain contains multiple immunoglobulin-binding domains, it may be, for example, a protein comprising the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0031] A polypeptide containing an immunoglobulin-binding domain of FpL may contain, for example, at its N- or C-terminus, an oligopeptide useful for specifically detecting or isolating a target substance. Examples of such oligopeptides include polyhistidine and polyarginine. Furthermore, the polypeptide may contain, for example, at its N- or C-terminus, an oligopeptide useful for immobilizing the polypeptide on a solid phase such as a chromatographic support. Examples of such oligopeptides include oligopeptides containing lysine or cysteine ​​residues.

[0032] In one embodiment, the method of the present invention comprises the steps of: culturing a recombinant Escherichia coli containing a polynucleotide encoding a polypeptide comprising at least the immunoglobulin-binding domain of FpL to express the polypeptide; and purifying the polypeptide from the culture of the recombinant Escherichia coli obtained in the expression step using chromatography, wherein the step of purifying the polypeptide comprises a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography. Each step is described in detail below.

[0033] Examples of Escherichia coli include Escherichia coli B strains such as the BL21(DE3) strain; the W3110 strain (ATCC 27325) and the MG1655 strain (ATCC 47076), and derivatives thereof.

[0034] The method for preparing a recombinant E. coli containing a polynucleotide encoding a polypeptide comprising at least the immunoglobulin-binding domain of FpL is not particularly limited, as long as the gene encoding the polypeptide comprising at least the immunoglobulin-binding domain of FpL is introduced into a host and the gene is retained in the host in an expressible manner.

[0035] Introduction of a gene into a host can be achieved by introducing the gene into the host's chromosome. Introduction of a gene into a chromosome can be achieved, for example, by using homologous recombination. Examples of gene introduction methods that utilize homologous recombination include methods that use a plasmid containing a temperature-sensitive replication origin.

[0036] Introduction of a gene into a host can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the target gene is ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the host.

[0037] The vector may be one capable of autonomous replication in host cells. The vector may also comprise a promoter and a terminator for expressing the inserted gene. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, and pCold vectors (all from Takara Bio Inc.), pACYC177, pACYC184, and pMW219 (all from Nippon Gene Co., Ltd.), pTrc99A (Pharmacia), pET vectors (Merck & Co.), and pQE vectors (Qiagen).

[0038] When a gene is introduced, it is sufficient that the gene can be expressed by the host. Specifically, it is sufficient that the gene is maintained so that it is expressed under the control of a promoter that functions in the host. A "promoter that functions in the host" may refer to a promoter that has promoter activity in the host. Promoters that function in the host are not particularly limited, and examples include the CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, tac promoter, trc promoter, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and the Pm1 promoter, PR promoter, PL promoter, P4 promoter, and P8 promoter derived from Bifidobacterium. The promoter that functions in the host may be a promoter derived from the host or a heterologous promoter.

[0039] By culturing a recombinant E. coli containing a polynucleotide encoding a polypeptide comprising at least the immunoglobulin-binding domain of FpL, the polypeptide can be expressed.

[0040] The step of expressing the polypeptide comprises: a step of culturing a genetically modified Escherichia coli containing an expression vector into which a polynucleotide encoding a polypeptide comprising an immunoglobulin-binding domain of FpL and an inducible promoter have been inserted (hereinafter also referred to as a culturing step); <ii>a step of adding an inducer when the cell concentration of the E. coli reaches a certain concentration, thereby expressing the polypeptide from the E. coli (hereinafter also referred to as an induction step); may include:

[0041] Culture process The medium used for culturing may be any medium that allows the E. coli to grow and allows expression of a polypeptide containing the immunoglobulin-binding domain of FpL. Examples of carbon sources include glucose, fructose, maltose, sucrose, raw sugar, and molasses, with glucose being preferred. Yeast extract is preferred as a nitrogen source, but polypeptone, casein and its metabolites, corn steep liquor, soy protein, meat extract, and fish extract may also be used. Examples of inorganic salts include sodium chloride, magnesium chloride, magnesium sulfate, iron(II) sulfate, iron(III) sulfate, iron(II) chloride, iron(III) chloride, iron citrate, ammonium iron sulfate, calcium chloride, calcium sulfate, zinc sulfate, zinc chloride, copper(II) sulfate, copper(II) chloride, manganese(II) sulfate, and manganese(II) chloride. Examples of vitamins include biotin, nicotinic acid, thiamine, riboflavin, inositol, and pyridoxine. However, if phosphate ions are added to the medium as the inorganic salt at the start of the culture, the growth of the Escherichia coli may be inhibited, and therefore, the medium does not necessarily need to contain phosphate ions. Note that the start of the culture may refer to the start of the main culture after the pre-culture.

[0042] <ii>induction process The inducer added to the culture medium may be selected appropriately depending on the promoter inserted into the expression vector. For example, when the promoter is a lac promoter or a tac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) can be used. The concentration of IPTG added is, for example, a final concentration of 0.005 mmol / L to 1.0 mmol / L, preferably a final concentration of 0.01 mmol / L to 0.5 mmol / L. Expression induction, such as IPTG induction, can be performed under conditions well known in the art.

[0043] Along with the inducer, phosphate ions can be added to the culture medium to a final concentration of 4 mmol / L to 50 mmol / L. Phosphate ion sources include phosphoric acid (H3PO4) and water-soluble phosphate salts such as disodium hydrogen phosphate (Na2HPO4), disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4), sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), trisodium phosphate dodecahydrate (Na3PO4·12H2O), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate trihydrate (K2HPO4), and dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O). The phosphate ion concentration may be within the aforementioned range, but is more preferably 5 mmol / L or more and 40 mmol / L or less, even more preferably 6 mmol / L or more and 30 mol / L or less, and even more preferably 6 mmol / L or more and 25 mol / L or less.

[0044] In the culturing and induction steps, recombinant E. coli can be cultured using either batch culture (also known as fed-batch culture) or perfusion culture, or a combination of these. However, adding nutrient sources such as carbon and nitrogen sources to the medium all at once at the start of culture can inhibit E. coli growth and protein expression, and can also produce by-products such as organic acids, potentially reducing protein expression efficiency. Therefore, it is preferable to culture recombinant E. coli using fed-batch culture, in which the amount of nutrient added at the start of culture is kept to a minimum and nutrient sources are supplied appropriately (fed-batch) during culture.

[0045] When culturing recombinant E. coli using fed-batch culture in the culturing and induction steps, the concentrations of the carbon and nitrogen sources added at the start of culturing are preferably 20 g / L or less if the carbon source is glucose, and 80 g / L or less if the nitrogen source is yeast extract. High-concentration solutions of the carbon and nitrogen sources are preferred because they can prevent an increase in the volume of the culture medium. Specifically, concentrations of 300 g / L to 900 g / L are preferred if the carbon source is glucose, and 100 g / L to 500 g / L are preferred if the nitrogen source is yeast extract. The inorganic salts mentioned above may also be added.

[0046] When culturing recombinant E. coli by fed-batch culture in the culture and induction steps, the carbon and nitrogen sources must be fed while maintaining the concentrations of the carbon and nitrogen sources in the medium at a predetermined low level. As used herein, "a predetermined low concentration" refers to a concentration at which the carbon source is not depleted and by-products such as organic acids are not produced. For example, when glucose is used as a carbon source, culturing at a carbon source concentration above 5 g / L may result in the accumulation of by-product organic acids, which may inhibit E. coli growth and the expression of the polypeptide. Therefore, the carbon source concentration should be at least 5 g / L or less, preferably 1 g / L or less, more preferably 0.5 g / L or less, and most preferably 0.1 g / L or less. There are no particular limitations on the method for monitoring carbon source depletion; for example, it can be monitored by a decrease in respiratory activity. A decrease in respiratory activity is manifested, for example, as an increase in the dissolved oxygen concentration (DO) of the culture medium, an increase in the oxygen concentration in the exhaust gas, a decrease in carbon dioxide concentration, or an increase in pH. In particular, DO is a preferred indicator for monitoring carbon source depletion because it responds quickly, as microbial respiratory activity decreases and then rises sharply when the carbon source is depleted.

[0047] As used herein, the culture of recombinant E. coli obtained in the expression step may refer to either the medium or the cells that have been subjected to appropriate treatment, or may refer to either the medium or the cells.

[0048] For example, a polypeptide containing the immunoglobulin-binding domain of FpL may be collected in the form contained in bacterial cells. The method for collecting bacterial cells is not particularly limited, and examples include spontaneous sedimentation, centrifugation, and filtration.

[0049] As another example, a polypeptide containing an immunoglobulin-binding domain of FpL may be recovered from the form contained in the bacterial cells by subjecting the bacterial cells to appropriate treatment. Such a recovered sample may be referred to as a "sample containing an immunoglobulin-binding domain (polypeptide) of FpL," as described below. Examples of bacterial cell treatments include immobilization on a carrier such as acrylamide or carrageenan, freeze-thawing, treatment to increase membrane permeability, and physical disruption using ultrasonic disruption or a pressure homogenizer. Membrane permeability can be increased, for example, by using a surfactant or organic solvent. These treatments may be used alone or in appropriate combination.

[0050] The step of purifying the polypeptide includes a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography. The anion exchange chromatography support used in the purification step using anion exchange chromatography is not particularly limited, but examples include TOYOPEARL SuperQ-650, TOYOPEARL GigaCapQ-650, TOYOPEARL DEAE-650, TOYOPEARL NH2-750, TOYOPEARL QAE-550, TOYOPEARL GigaCap DEAE-650, TOYOPEARL Q-600C AR (all manufactured by Tosoh Corporation), Q Sepharose Fast Flow (manufactured by Cytiva), and Eshmuno Q (manufactured by Merck), and the particularly strong ion exchangers TOYOPEARL SuperQ-650, TOYOPEARL GigaCapQ-650, TOYOPEARL Q-600C AR, Q Sepharose Fast Flow, and Eshmuno Q are more preferred in that they improve the purification purity of a polypeptide containing at least the immunoglobulin-binding domain of FpL.

[0051] The hydrophobic chromatography support used in the purification step using hydrophobic chromatography is not particularly limited, but examples include TOYOPEARL Butyl-600, TOYOPEARL Phenyl-600, TOYOPEARL PPG-600, TOYOPEARL Ether-650, TOYOPEARL SuperButyl-550, TOYOPEARL Hexyl-650 (all manufactured by Tosoh Corporation), Phenyl Sepharose 6 Fast Flow (manufactured by Cytiva), and Fractogel Phenyl (manufactured by Merck). In particular, TOYOPEARL Butyl-600, TOYOPEARL Phenyl-600, TOYOPEARL SuperButyl-550, Phenyl Sepharose 6 Fast Flow, and Fractogel Phenyl, in which the functional group of the support is a butyl group or a phenyl group, are more preferred in terms of improving the purification purity of a polypeptide comprising at least the immunoglobulin-binding domain of FpL.

[0052] The purification step using anion exchange chromatography may be followed by the purification step using hydrophobic chromatography, or vice versa. Another step may be added before, after, or between the purification step using anion exchange chromatography and the purification step using hydrophobic chromatography.

[0053] The purification process using hydrophobic chromatography includes the steps of equilibrating a hydrophobic chromatography column, applying to the column a sample containing a polypeptide that contains at least the immunoglobulin-binding domain of Protein L (FpL) derived from bacteria of the genus Finegoldia, and recovering the polypeptide.

[0054] Column equilibration refers to the process of pumping a solution into the column at a volume 1 to 10 times or more than 10 times the column volume.

[0055] At least one of the solution for equilibrating the column (also referred to as the equilibration solution) and the sample containing a polypeptide containing at least the immunoglobulin-binding domain of Finegoldia Protein L (FpL) contains 0.4 mol / L to 1.1 mol / L of ammonium sulfate. More preferably, at least one of the equilibration solution and the sample further contains sodium chloride.

[0056] There may or may not be a pH change between the purification step using anion exchange chromatography and the purification step using hydrophobic chromatography. Since there is no pH change between the purification step using anion exchange chromatography and the purification step using hydrophobic chromatography, there is no possibility of purifying the target protein across its isoelectric point, which is preferable in that any form of Protein L can be applied to its purification.

[0057] "No pH change" between the anion exchange chromatography purification step and the hydrophobic chromatography purification step may mean, for example, that hydrophobic chromatography is performed by applying the eluate to a hydrophobic chromatography support equilibrated with an equilibration solution having a pH equivalent to that of the eluate eluted from the anion exchange support. "No pH change" between the anion exchange chromatography purification step and the hydrophobic chromatography purification step may mean, for example, that anion exchange chromatography is performed by applying the eluate to an anion exchange support equilibrated with an equilibration solution having a pH equivalent to that of the eluate eluted from the hydrophobic chromatography support. The "equivalent pH" may mean that the difference in pH between the eluate and the equilibration solution is 1.0 or less, 0.5 or less, 0.3 or less, or 0.1 or less. [Example]

[0058] The present invention will be explained in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0059] Example 1 Preparation of recombinant E. coli culture A culture of recombinant Escherichia coli capable of expressing a polypeptide containing at least the immunoglobulin-binding domain of Protein L (hereinafter also referred to as "FpL") derived from the genus Finegoldia was prepared by the method described below.

[0060] (1) The Escherichia coli BL21(DE3) strain was transformed with expression vectors containing polynucleotides encoding (FpL_C3KX 7e)4-IT-3K (SEQ ID NO: 5), (FpL_C3KX 9a)4-IT-3K (SEQ ID NO: 6), and (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7), which are polypeptides containing at least the immunoglobulin-binding domain of FpL, and an inducible promoter, to produce recombinant Escherichia coli capable of expressing these polypeptides.

[0061] (FpL_C3KX 7e)4-IT-3K (SEQ ID NO: 5) is a polypeptide in which four FpL_C3KX 7e (SEQ ID NO: 2) amino acid substitutions of the C3 domain of FpL (amino acid residues 394 to 463 of GenBank No. AAA67503, SEQ ID NO: 1, hereinafter also referred to as "FpL_C3") are directly linked together, and an oligopeptide (SEQ ID NO: 4) and three lysine residues are added to the C-terminus to improve the immobilization rate to an insoluble carrier. (FpL_C3KX 9a)4-IT-3K (SEQ ID NO: 6) is a polypeptide in which four FpL_C3KX 9a (SEQ ID NO: 3), which are amino acid substitutions of FpL_C3 (SEQ ID NO: 1), are linked together, and an oligopeptide having the sequence set forth in SEQ ID NO: 4 and three lysine residues are added to the C-terminus of the FpL_C3KX 9a. (FpL_C3KX 9a)5-IT-3K (sequence number 7) is a polypeptide consisting of five FpL_C3KX 9a (sequence number 3), which are amino acid substitutions of FpL_C3 (sequence number 1), linked together, and an oligopeptide consisting of the sequence set forth in sequence number 4 and three lysine residues added to the C-terminus.

[0062] Furthermore, the FpL_C3KX 7e (SEQ ID NO: 2) is a polypeptide into which the amino acid substitutions E4G (this description indicates that the fourth glutamic acid in SEQ ID NO: 1 has been substituted with glycine; the same applies hereinafter), P6S, K7A, K13R, K22R, K29I, K38E, K48R, E49D, N50Y, Y53F, N62Y, K67R, and A69V have been introduced into FpL_C3 (SEQ ID NO: 1) (JP 2023-064059 A), The FpL_C3KX 9a (SEQ ID NO: 3) is a polypeptide in which the amino acid substitutions E4G, P6S, K7A, K13R, K22R, I23R, K29I, K38E, N44R, K48R, E49D, N50Y, Y53F, N62Y, K67R and A69V have been introduced into FpL_C3 (SEQ ID NO: 1) (JP 2023-103953 A).

[0063] (2) The recombinant E. coli prepared in (1) was inoculated into 50 mL of 2xYT liquid medium (1.6% (w / v) Phyton peptone (Thermo Fisher Scientific), 1% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing 50 μg / mL kanamycin placed in a 250 mL baffled flask, and pre-cultured at 30°C for 23 hours.

[0064] (3) 36 mL of the preculture solution from (2) was added to a 3 L culture tank containing 1.2 L of autoclave-sterilized main culture medium (4% (w / v) yeast extract, 0.24% (w / v) magnesium sulfate heptahydrate, 0.05% (w / v) sodium chloride, 0.0186% (w / v) potassium chloride, 2% (w / v) glucose, 50 μg / mL kanamycin), and main culture was started under the following conditions: temperature 30°C, agitation speed 400 rpm, pH 6.9 to 7.1, aeration rate (air) 1.5 L / min, and the agitation speed (maximum 700 rpm) was automatically controlled so that the dissolved oxygen concentration was approximately 40% of the saturation concentration. When the glucose in the medium was consumed and depleted, a feed medium (42.5% (w / v) glucose, 14.2% (w / v) yeast extract, 1.2% (w / v) magnesium sulfate heptahydrate) was added using the DO stat method.

[0065] (4) Sixteen hours after the start of the main culture, 1.5 mL of 0.1 mol / L IPTG (Isopropyl-β-D-thiogalactopyranoside) was added, and the culture was continued for another 24 hours. The resulting culture medium was centrifuged, and E. coli cells containing immunoglobulin-binding proteins were collected.

[0066] (5) The polypeptide expressed in the bacterial cells was extracted by adding 4 mL of an extraction solution (50 mmol / L sodium phosphate buffer (pH 7.5) containing 1 mmol / L EDTA (pH 8.0), 2 mmol / L magnesium sulfate, 250 units / L endonuclease (Kaneka), 0.005% (w / v) lysozyme, and 0.5% (w / v) Triton X-100 (Merck)) to 1 g (wet weight) of the bacterial cells recovered in (4).

[0067] (6) The bacterial extract was centrifuged, the supernatant was collected, and then filtered through a 0.2 μm filter to obtain samples containing a polypeptide containing at least the immunoglobulin-binding domain of FpL (hereinafter simply referred to as "FpL polypeptide"), which was the target of purification by chromatography (culture from recombinant E. coli).

[0068] Example 2 Purification using a hydrophobic chromatography column (1) To a 5.2 mL sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) from the purification target obtained in Example 1, 0.9 mol / L ammonium sulfate (hereinafter also abbreviated as "ammonium sulfate") and 0.4 mol / L sodium chloride were added, followed by stirring at room temperature for 3 hours. After stirring, the supernatant was recovered by centrifugation (recovered supernatant: 7.8 mL).

[0069] (2) 7.8 mL of the supernatant collected in (1) was applied to a column (hydrophobic chromatography column) packed with 1 mL of hydrophobic chromatography support, which had been pre-equilibrated with 50 mmol / L sodium phosphate buffer (pH 7.5) containing 0.9 mol / L ammonium sulfate and 0.6 mol / L sodium chloride (hereinafter also referred to as "equilibration solution A"), to adsorb (FpL_C3KX 9a)5-IT-3K to the support. The hydrophobic chromatography support used was one of the following (both manufactured by Tosoh Corporation): H1: TOYOPEARL Butyl-600M (hereinafter referred to simply as "Butyl") H2: TOYOPEARL Phenyl-600M (hereinafter simply referred to as "Phenyl") H3: TOYOPEARL PPG-600M (hereinafter referred to simply as "PPG"). Furthermore, the protein concentration in the supernatant was measured using the method described below and was found to be 10.7 mg / mL, which means that the load on the column was 83.5 mg / mL (carrier).

[0070] (3) After a pre-elution washing step using 5 mL of equilibration solution A to wash away impurities from the hydrophobic chromatography column, 5 mL of 50 mmol / L sodium phosphate buffer (pH 7.5) (hereinafter also referred to as "buffer A") was used to elute (FpL_C3KX 9a)5-IT-3K adsorbed to the hydrophobic chromatography support, and 5 mL of a fraction containing the polypeptide was collected.

[0071] (4) The fractions collected in (3) were subjected to size exclusion chromatography (TSKgel G2000SWXL, Tosoh Corporation) and SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The purity of (FpL_C3KX 9a)5-IT-3K was confirmed by measuring the peak area percentage of the peak corresponding to (FpL_C3KX 9a)5-IT-3K.

[0072] (5) The supernatant collected in (1) and the fractions collected in (3) were measured for absorbance at 280 nm using a NanoDrop One (Thermo Fisher Scientific). The protein concentration calculated from the absorbance, the volume of the solution, and the purity measured in (4) were used to calculate the recovery amount of (FpL_C3KX 9a)5-IT-3K purified using various hydrophobic chromatography columns.

[0073] The results of SDS-PAGE of the fractions collected in (3) are shown in Figure 1, and the results of purification are shown in Table 1. Regardless of the hydrophobic chromatography column used, a band corresponding to (FpL_C3KX 9a)5-IT-3K (shown by the white arrow in Figure 1) was observed in the eluted fractions. Compared to the (FpL_C3KX 9a)5-IT-3K band in the sample before chromatographic purification, the band after purification was darker, indicating that the purity of (FpL_C3KX 9a)5-IT-3K was improved by purification.

[0074] These results demonstrate that the FpL polypeptide contained in the sample can be purified using a hydrophobic chromatography column. Furthermore, when the purity of (FpL_C3KX 9a)5-IT-3K (referred to as "FpL" in Table 1) contained in the fractions recovered in (3) was compared, it was found that the purity was improved when butyl or phenyl was used as the hydrophobic chromatography support compared to when PPG was used (Table 1). Furthermore, it was found that the amount of FpL recovered was particularly high when phenyl was used as the hydrophobic chromatography support.

[0075] [Table 1]

[0076] Example 3 Purification using an anion exchange chromatography column (1) (FpL_C3KX 9a)5-IT-3K, one of the purified targets obtained in Example 1 Five mL of a sample containing (SEQ ID NO: 7) was applied to a column (anion exchange chromatography column) packed with 1 mL of anion exchange chromatography support, which had been previously equilibrated with buffer A (50 mmol / L sodium phosphate buffer (pH 7.5)), to adsorb (FpL_C3KX 9a)5-IT-3K onto the support. The anion exchange chromatography support used was one of the following (all manufactured by Tosoh Corporation): A1: TOYOPEARL SuperQ-650M (hereinafter referred to simply as "SuperQ") A2: TOYOPEARL GigaCapQ-650M (hereinafter referred to simply as "GigaCapQ") A3: TOYOPEARL DEAE-650M (hereinafter referred to simply as "DEAE"). Furthermore, the protein concentration in the sample was measured by the method described in Example 2(5) and was found to be 16.6 mg / mL, so the load on the column was 83.0 mg / mL (carrier).

[0077] (2) After a pre-elution wash step using 5 mL of buffer A to wash away impurities from the anion exchange chromatography column, (FpL_C3KX 9a)5-IT-3K adsorbed on the anion exchange chromatography support was eluted using 5 mL of buffer A containing 1 mol / L sodium chloride, and fractions containing the polypeptide were collected.

[0078] (3) The purity of (FpL_C3KX 9a)5-IT-3K contained in the fractions recovered in (2) was confirmed by the method described in Example 2(4), and the recovery amount of (FpL_C3KX 9a)5-IT-3K purified using various anion exchange chromatography columns was calculated from the protein concentration measured by the method described in Example 2(5).

[0079] Figure 1 shows the SDS-PAGE results for the fractions collected in (2), and Table 2 shows the purification results. Regardless of the anion-exchange chromatography column used, a band corresponding to (FpL_C3KX 9a)5-IT-3K (shown by the white arrow in Figure 1) was observed in the eluted fractions. Compared to the (FpL_C3KX 9a)5-IT-3K band in the sample before chromatographic purification, the band after purification was darker, indicating that the purity of (FpL_C3KX 9a)5-IT-3K was improved by purification.

[0080] These results demonstrate that the FpL polypeptide contained in the sample can be purified using an anion exchange chromatography column. Furthermore, when the purity of (FpL_C3KX 9a)5-IT-3K (referred to as "FpL" in Table 2) contained in the fractions collected in (2) was compared, it was found that the purity was improved when SuperQ or GigaCapQ was used as the anion exchange chromatography support compared to when DEAE was used as the anion exchange chromatography support (Table 2). Furthermore, it was found that the amount of FpL recovered was greater when SuperQ or GigaCapQ was used as the anion exchange chromatography support compared to when DEAE was used as the anion exchange chromatography support.

[0081] [Table 2]

[0082] Comparative Example 1 Purification using a cation exchange chromatography column (1) 5 mL of a sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7), one of the targets for purification obtained in Example 1, was applied to a column (cation exchange chromatography column) packed with a cation exchange chromatography support (volume: 1 mL) pre-equilibrated with 50 mmol / L sodium phosphate buffer (pH 6.0) (hereinafter also referred to as "Buffer B"), and the flow-through fraction was collected. The cation exchange chromatography support used was one of the following (all manufactured by Tosoh Corporation): C1: TOYOPEARL SP-650M (hereinafter referred to simply as "SP") C2: TOYOPEARL CM-650M (hereinafter referred to simply as "CM") C3: TOYOPEARL GigaCapS-650M (hereinafter referred to simply as "GigaCapS") Furthermore, the protein concentration in the sample was measured by the method described in Example 2(5) and was found to be 16.6 mg / mL, so the load on the column was 83.0 mg / mL (carrier).

[0083] (2) A washing step was carried out using 5 mL of buffer solution B to wash away impurities in the cation exchange chromatography column, and a pre-elution wash fraction was collected.

[0084] (3) Proteins adsorbed on various cation exchange chromatography carriers were eluted using 5 mL of buffer B containing 1 mol / L sodium chloride, and the eluted fractions were collected.

[0085] (4) The flow-through fraction collected in (1), the wash fraction collected in (2), and the elution fraction collected in (3) were each subjected to SDS-PAGE to identify proteins not adsorbed to various cation exchange chromatography supports (flow-through fraction, wash fraction) and proteins adsorbed to various cation exchange chromatography supports (elution fraction).

[0086] The SDS-PAGE results for each fraction are shown in Figure 2. Regardless of the cation exchange chromatography column used, the band corresponding to (FpL_C3KX 9a)5-IT-3K (white arrow in Figure 2) was observed in the flow-through fractions (C1T, C2T, and C3T) and the wash fractions (lanes C1W, C2W, and C3W in Figure 2), but not in the elution fractions (lanes C1E, C2E, and C3E in Figure 2). These results demonstrate that the FpL polypeptide contained in the sample cannot be purified using a cation exchange chromatography column.

[0087] Example 4 Purification using an anion exchange chromatography column (salt concentration study) (1) 12 mL of a sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) among the purification targets obtained in Example 1 was applied to a column (SuperQ column) packed with SuperQ (volume: 1 mL) that had been equilibrated in advance with 50 mmol / L Tris-HCl buffer (pH 7.5) (hereinafter also referred to as "Buffer C") containing any of the salts shown in (a) to (d) below, to adsorb the immunoglobulin-binding protein onto the carrier: (a) 50 mmol / L sodium chloride (b) 100 mmol / L sodium chloride (c) 150 mmol / L sodium chloride (d) 200 mmol / L sodium chloride. The protein concentration in the sample was measured by the method described in Example 2(5) and was found to be 12.5 mg / mL, so the load on the column was 150 mg / mL (carrier).

[0088] (2) After a pre-elution wash step using 8 mL of the same buffer solution used for equilibration in (1) to wash away impurities from the SuperQ column, (FpL_C3KX 9a)5-IT-3K adsorbed to the SuperQ column was eluted using 5 mL of buffer solution C containing 250 mmol / L sodium chloride, and fractions containing the polypeptide were collected.

[0089] (3) A washing step was carried out using 5 mL of buffer C containing 1 mol / L sodium chloride to wash away proteins remaining in the SuperQ column.

[0090] (4) The fractions collected in (2) were subjected to size exclusion chromatography (column used: TSKgel G2000SW XL The purity of (FpL_C3KX 9a)5-IT-3K contained in the fraction was measured by subjecting the fraction to a SuperQ column (Tosoh Corporation). The recovery amount of (FpL_C3KX 9a)5-IT-3K purified using the SuperQ column was calculated from the protein concentration measured by the method described in Example 2(5).

[0091] Figure 3 shows the effect of different salt concentrations in the buffer used for equilibration on the chromatogram, and Table 3 shows the purification results. A peak corresponding to (FpL_C3KX 9a)5-IT-3K (shown by the white arrow in Figure 3) was observed using both buffers. Furthermore, when the yield of (FpL_C3KX 9a)5-IT-3K (referred to as "FpL" in Table 3) contained in the fractions collected in (2) was compared, it was found that when the sodium chloride concentration in the buffer used for equilibration was between 30 mmol / L and 170 mmol / L, the yield was higher than when it was 180 mmol / L or higher (Table 3). Furthermore, when the purity of the polypeptide contained in the fractions collected in (2) was compared, it was found that when the sodium chloride concentration in the buffer used for equilibration was between 70 mmol / L and 170 mmol / L, the purity was higher than when it was 60 mmol / L or lower or 180 mmol / L or higher (Table 3).

[0092] [Table 3]

[0093] Example 5 Purification using a hydrophobic chromatography column (investigation of ammonium sulfate concentration) (1) To a 5 mL sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) among the purification targets obtained in Example 1, ammonium sulfate was added in the amount shown in any of (a) to (e) below, and the mixture was stirred at room temperature for 3 hours. (a) 0.3 mol / L (b) 0.6 mol / L (c) 0.9 mol / L (d) 1.2 mol / L (e) 1.5 mol / L After stirring, the supernatant was collected by centrifugation. The resulting supernatant volumes were 5.6 mL for (a), 6.3 mL for (b), 7.1 mL for (c), 8.3 mL for (d), and 10 mL for (e).

[0094] (2) The precipitate obtained after collecting the supernatant in (1) was suspended in 5 mL of phosphate buffered saline (PBS, pH 7.4). The supernatant collected in (1) and the resulting suspension were subjected to SDS-PAGE to confirm the presence of (FpL_C3KX 9a)5-IT-3K in the supernatant and the resulting suspension.

[0095] (3) The entire supernatant collected in (1) was applied to a column (phenyl column) packed with 1 mL of phenyl, which had been pre-equilibrated with buffer A containing ammonium sulfate at the concentration indicated in any one of (a) to (e), to adsorb (FpL_C3KX 9a)5-IT-3K to the phenyl. The protein concentrations in the supernatants were measured by the method described in Example 2(5), and were found to be 14.9 mg / mL for (a), 12.4 mg / mL for (b), 10.7 mg / mL for (c), 7.8 mg / mL for (d), and 4.5 mg / mL for (e), respectively. Therefore, the loading amount onto the column is 82.7 mg / mL (carrier) for (a), 77.5 mg / mL (carrier) for (b), 76.4 mg / mL (carrier) for (c), 65.0 mg / mL (carrier) for (d), and 45.0 mg / mL (carrier) for (e).

[0096] (4) A pre-elution wash step was performed to wash away impurities from the phenyl column using 5 mL of the buffer solution used for equilibration in (3). Then, 5 mL of buffer solution A was used to elute (FpL_C3KX 9a)5-IT-3K adsorbed to the phenyl column, and fractions containing the polypeptide were collected.

[0097] (5) Using a method similar to that used in Example 4(4), the purity of (FpL_C3KX 9a)5-IT-3K contained in the fractions recovered in (4) was measured, and the recovery amount of (FpL_C3KX 9a)5-IT-3K purified using a phenyl column was calculated.

[0098] The results of SDS-PAGE of the supernatant collected in (1) and the precipitate suspension prepared in (2) are shown in FIG. 4, and the results of purification are shown in Table 4.

[0099] When the ammonium sulfate concentration added to the sample and equilibration buffer was 1.2 mol / L or higher, (FpL_C3KX 9a)5-IT-3K partially precipitated due to the salting-out effect, and a band corresponding to (FpL_C3KX 9a)5-IT-3K was observed on the precipitated side (Figure 4, white arrow). This indicates a decrease in the recovery of the polypeptide. Furthermore, the purity of (FpL_C3KX 9a)5-IT-3K (referred to as "FpL" in Table 4) contained in the fractions recovered in (3) was also significantly reduced (Table 4). Furthermore, Table 4 shows that when the ammonium sulfate concentration added to the sample and equilibration buffer was 0.3 mol / L or lower, the amount of (FpL_C3KX 9a)5-IT-3K recovered in the elution fractions was reduced due to a decrease in the amount of adsorption to the hydrophobic chromatography gel. Therefore, it was found that when the ammonium sulfate concentration added to the sample and the buffer solution used for equilibration was 0.4 mol / L or more and 1.1 mol / L or less, the FpL purity was maintained at 40% or more while a large amount of FpL was recovered.

[0100] [Table 4]

[0101] Example 6 Purification using a hydrophobic chromatography column (examination of mixed salts) (1) (FpL_C3KX 9a)5-IT-3K, one of the purified targets obtained in Example 1 After adding the amount of salt shown in any one of (a) to (c) below to a sample containing (SEQ ID NO: 7), The mixture was stirred at room temperature for 3 hours. (a) 0.8 mol / L ammonium sulfate (sample volume 10.0 mL) (b) 0.8 mol / L ammonium sulfate and 1 mol / L sodium chloride (sample volume: 21.9 mL) (c) 0.8 mol / L ammonium sulfate and 2 mol / L sodium chloride (sample volume: 28.3 mL) were stirred and centrifuged to recover the supernatant. The supernatant volumes obtained were 13.6 mL for (a), 41 mL for (b), and 85 mL for (c), respectively.

[0102] (2) The entire supernatant collected in (1) was applied to a column (butyl column) packed with butyl (volume: 1 mL) pre-equilibrated with buffer A (50 mmol / L sodium phosphate buffer, pH 7.5) containing the salt concentration indicated in any of (a) to (c) above, allowing (FpL_C3KX 9a)5-IT-3K to be adsorbed onto the butyl. The protein concentrations in the supernatant were measured as described in Example 2(5), and were found to be 8.4 mg / mL for (a), 5.5 mg / mL for (b), and 2.5 mg / mL for (c). Therefore, the column loads were 114 mg / mL (carrier) for (a), 226 mg / mL for (b), and 213 mg / mL for (c), respectively.

[0103] (3) A pre-elution washing step was carried out to wash away impurities from the hydrophobic chromatography column using 10 mL of the buffer solution used for equilibration in (2).

[0104] (4) When equilibration was performed with buffer A containing (a) above, 5 mL of buffer A containing 0.1 mol / L ammonium sulfate was applied; when equilibration was performed with buffer A containing (b) above, 5 mL of buffer A containing 0.04 mol / L ammonium sulfate and 0.05 mol / L sodium chloride was applied; and when equilibration was performed with buffer A containing (c) above, 5 mL of buffer A containing 0.04 mol / L ammonium sulfate and 0.1 mol / L sodium chloride was applied. This eluted (FpL_C3KX 9a)5-IT-3K adsorbed to Butyl, and fractions containing the polypeptide were recovered.

[0105] (5) A washing step was carried out using 4 mL of buffer solution A to wash away proteins remaining in the hydrophobic chromatography column.

[0106] (6) Using a method similar to that used in Example 4(4), the purity of (FpL_C3KX 9a)5-IT-3K contained in the fractions recovered in (4) was measured, and the recovery amount of (FpL_C3KX 9a)5-IT-3K purified using a Butyl column was calculated.

[0107] Figure 5 shows the results of comparing the effects on the chromatogram due to differences in the buffer solution used for equilibration. The results of the purification are shown in Table 5. A peak corresponding to (FpL_C3KX 9a)5-IT-3K (shown by the white arrow in Figure 5) was observed regardless of the buffer used. Furthermore, a comparison of the yield of (FpL_C3KX 9a)5-IT-3K (referred to as "FpL" in Table 5) contained in the fractions collected in (4) revealed that the yield was improved when a buffer containing ammonium sulfate and sodium chloride was used as the equilibration buffer compared to when a buffer containing only ammonium sulfate was used (Table 5).

[0108] [Table 5]

[0109] Example 7 Purification using a combination of an anion exchange chromatography column and a hydrophobic chromatography column (part 1) We investigated whether purification using an anion exchange chromatography column followed by purification using a hydrophobic chromatography column would improve the purity of the purified product compared to purification using only an anion exchange chromatography column or a hydrophobic chromatography column.

[0110] (1) First-stage purification (purification using an anion exchange chromatography column) (1-1) The sample to be purified (the sample containing the FpL polypeptide) obtained in Example 1 was applied to a column (anion exchange chromatography column) packed with 5 mL of anion exchange chromatography support pre-equilibrated with buffer C (50 mmol / L Tris-HCl buffer (pH 7.5) containing sodium chloride), and the polypeptide was adsorbed onto the support. Table 6 summarizes the FpL polypeptide, the amount of sample applied, the protein concentration measured by the method described in Example 2(5), the column used and the load amount onto the column, and the sodium chloride concentration contained in buffer C under each of the conditions (conditions (a) to (d)) examined in this example. In Table 6, FpL polypeptide is simply referred to as FpL.

[0111] [Table 6]

[0112] (1-2) A pre-elution wash step was performed to wash away impurities from the column using the buffer solution used to equilibrate the anion exchange chromatography column (conditions (a) to (c): 40 mL, condition (d): 60 mL). Then, the FpL polypeptide adsorbed on the anion exchange chromatography support was eluted using buffer solution C containing sodium chloride at the concentration shown in Table 6 (conditions (a) to (c): 25 mL, condition (d): 30 mL), and fractions containing the polypeptide were collected.

[0113] (1-3) A washing step was carried out using 25 mL of buffer C containing 1 mol / L sodium chloride to wash away proteins remaining in the anion exchange chromatography column.

[0114] The chromatograms obtained under each condition in the first purification step are shown in Figure 6. A peak corresponding to the FpL polypeptide (white arrow in Figure 6) was confirmed under all conditions.

[0115] (2) Second-stage purification (purification using a hydrophobic chromatography column) (2-1) Ammonium sulfate was added to the fractions collected in (1-2), and the mixture was stirred at room temperature for 3 hours. The supernatant was then centrifuged to recover the supernatant. Table 7 shows the amount of ammonium sulfate added, the volume of the supernatant obtained, and the protein concentration in the supernatant measured by the method described in Example 2(5) under each of the conditions (conditions (a) to (d)) examined in this example.

[0116] [Table 7]

[0117] (2-2) The entire supernatant obtained in (2-1) was applied to a column (hydrophobic chromatography column) packed with 5 mL of a hydrophobic chromatography support that had been pre-equilibrated with Buffer A (50 mmol / L sodium phosphate buffer, pH 7.5) containing ammonium sulfate, allowing the FpL polypeptide to adsorb onto the support. The ammonium sulfate concentrations in the equilibration solution and the load on the hydrophobic chromatography column under each condition are shown in Table 7.

[0118] (2-3) After a pre-elution wash step in which impurities in the hydrophobic chromatography column were washed away using 25 mL of the buffer solution used to equilibrate the hydrophobic chromatography column, the FpL polypeptide adsorbed to the hydrophobic chromatography carrier was eluted using 25 mL of buffer solution A containing ammonium sulfate at the concentration shown in Table 7, and fractions containing the polypeptide were collected.

[0119] (2-4) A washing step was carried out using 20 mL of buffer A to wash away proteins remaining in the hydrophobic chromatography column.

[0120] (2-5) Using a method similar to that described in Example 4(4), the purity of the FpL polypeptide contained in the fractions recovered in (1-2) and (2-3) was measured, and the amount of FpL polypeptide purified using each chromatographic column was calculated.

[0121] Figure 7 shows the chromatograms obtained under each condition in the second purification step. A peak corresponding to the FpL polypeptide (white arrow in Figure 7) was observed under all conditions, and the peak shape was sharper than the corresponding peak in the first purification step (white arrow in Figure 6). The horizontal axis of the chromatogram represents the liquid volume, so the sharper the peak shape, the smaller the amount of solution used. As the purification scale increases, the greater the amount of liquid handled, the greater the workload, so it is desirable to recover the protein using as little solution as possible. Furthermore, a smaller amount of solution allows for the recovery of protein at a higher concentration, which is thought to reduce the labor required for the concentration step.

[0122] The purification results under each condition in this example are shown in Table 8. The purity of the FpL polypeptide contained in the elution fraction after the second purification step was 90% or higher under all conditions, which was improved compared to purification using only an anion exchange chromatography column (Examples 3 and 4) or a hydrophobic chromatography column (Examples 2, 4, and 5). These results demonstrate that when purifying the FpL polypeptide from a culture of recombinant E. coli capable of expressing the polypeptide using chromatography, the polypeptide can be purified to a higher purity by including a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography. It also demonstrates that the polypeptide can be purified to a higher purity even when the pH is not changed between the purification using the anion exchange chromatography column and the purification using the hydrophobic chromatography column.

[0123] [Table 8]

[0124] Example 8 Purification using a combination of an anion exchange chromatography column and a hydrophobic chromatography column (part 2) An investigation was conducted to determine whether the purification method of this embodiment is applicable even when the column size is scaled up (that is, even in large-scale purification).

[0125] (1) First-stage purification (purification using an anion exchange chromatography column) (1-1) A column packed with SuperQ (volume: 100 mL) was used as the anion exchange chromatography column (SuperQ column). The polypeptide was adsorbed onto SuperQ in the same manner as in Example 7(1-1), except that the FpL polypeptide, the amount of sample applied, the protein concentration measured by the method described in Example 2(5), and the sodium chloride concentration in Buffer C under each condition (conditions (a) and (b)) were as shown in Table 9. In Table 9, FpL polypeptide is simply referred to as FpL. The load amount onto the SuperQ column is also shown in Table 9.

[0126] [Table 9]

[0127] (1-2) A pre-elution wash step was performed to wash away impurities from the column using the buffer solution used to equilibrate the SuperQ column (condition (a): 800 mL, condition (b): 1200 mL). After that, the FpL polypeptide adsorbed to SuperQ was eluted using buffer solution C containing 300 mmol / L sodium chloride (condition (a): 500 mL, condition (d): 600 mL), and fractions containing the polypeptide were collected.

[0128] (1-3) A washing step was carried out using 400 mL of buffer C containing 1 mol / L sodium chloride to wash away proteins remaining in the SuperQ column.

[0129] (2) Second-stage purification (purification using a hydrophobic chromatography column) (2-1) Ammonium sulfate was added to the fractions collected in (1-2), and the mixture was stirred at room temperature for 3 hours. The supernatant was then collected by centrifugation. Table 10 summarizes the amount of ammonium sulfate added, the volume of the supernatant obtained, and the protein concentration in the supernatant measured by the method described in Example 2(5) under each of the conditions (conditions (a) and (b)) examined in this example.

[0130] [Table 10]

[0131] (2-2) The hydrophobic chromatography column was a column packed with butyl (volume: 100 mL) (butyl column), and the polypeptide was adsorbed onto butyl in the same manner as in Example 7(2-2), except that the ammonium sulfate concentration in buffer A (50 mmol / L sodium phosphate solution (pH 7.5)) under each condition (conditions (a) and (b)) was set to the value shown in Table 10. In Table 10, the FpL polypeptide is simply referred to as FpL. The load amount onto the butyl column is also shown in Table 10.

[0132] (2-3) A pre-elution wash step was performed in which impurities in the Butyl column were washed away using 500 mL of the buffer solution used to equilibrate the Butyl column. Then, the FpL polypeptide adsorbed to the Butyl column was eluted using 500 mL of buffer solution A containing ammonium sulfate at the concentration shown in Table 10, and fractions containing the polypeptide were collected.

[0133] (2-4) A washing step was carried out using 400 mL of buffer A to wash away proteins remaining in the hydrophobic chromatography column.

[0134] (2-5) In the same manner as in Example 4(4), the purity of the FpL polypeptide contained in the fractions recovered in (1-2) and (2-3) was measured, and the recovered amount of the polypeptide was calculated.

[0135] The chromatograms obtained under each condition in this example are shown in Figure 8. Under all conditions and at all purification stages, a peak corresponding to the FpL polypeptide (white arrow in Figure 8) was confirmed.

[0136] The purification results under each condition in this example are shown in Table 11. The purity of the FpL polypeptide contained in the elution fraction after the second purification step was 90% or higher under all conditions, demonstrating that the purification method of this embodiment can be applied to large-scale purification.

[0137] [Table 11]

[0138] Example 9 Purification using a combination of an anion exchange chromatography column and a hydrophobic chromatography column (part 3) In Examples 7 and 8, purification using an anion exchange chromatography column was performed followed by purification using a hydrophobic chromatography column, but an investigation was conducted to see whether the effect of improving purity could be obtained even if the purification order was reversed.

[0139] (1) First-stage purification (purification using a hydrophobic chromatography column) (1-1) Ammonium sulfate and sodium chloride were added to a sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) from the purification target obtained in Example 1, and the mixture was stirred at room temperature for 3 hours, after which the supernatant was collected by centrifugation. Table 12 summarizes the sample volume, the amount of ammonium sulfate and sodium chloride added, and the protein concentration in the supernatant measured by the method described in Example 2(5) under each of the conditions (conditions (a) and (b)) examined in this example.

[0140] [Table 12]

[0141] (1-2) The supernatant prepared in (1-1) was applied to a phenyl-packed column (phenyl column) pre-equilibrated with buffer A containing ammonium sulfate and sodium chloride, allowing (FpL_C3KX 9a)5-IT-3K to be adsorbed onto the phenyl. The volume of the phenyl column and the concentrations of ammonium sulfate and sodium chloride contained in the equilibration solution under each condition are shown in Table 12. The load amount onto the phenyl column is also shown in Table 12.

[0142] (1-3) A pre-elution wash step was performed to wash away impurities from the phenyl column using the buffer solution used to equilibrate the phenyl column (condition (a): 60 mL, condition (b): 1200 mL). After that, the (FpL_C3KX 9a)5-IT-3K adsorbed to the phenyl column was eluted using buffer solution A (condition (a): 30 mL, condition (b): 650 mL), and fractions containing the polypeptide were collected.

[0143] (2) Second-stage purification (purification using an anion exchange chromatography column) (2-1) The fractions collected in (1-3) were diluted two-fold with buffer solution D (50 mmol / L sodium phosphate buffer solution (pH 6.5)) containing 0.025% (w / v) Tween 20 (trade name, manufactured by Tokyo Chemical Industry Co., Ltd.).

[0144] (2-2) The sample diluted in (2-1) was applied to a column packed with SuperQ (SuperQ column) pre-equilibrated with buffer D containing 200 mmol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name), allowing (FpL_C3KX 9a)5-IT-3K to be adsorbed onto the SuperQ. The SuperQ column capacity, sodium chloride concentration in the equilibration solution, and protein concentration in the sample measured by the method described in Example 2(5) under each condition are shown in Table 13. The load amount onto the SuperQ column is also shown in Table 13.

[0145] [Table 13]

[0146] (2-3) A pre-elution wash step was performed using Buffer D (condition (a): 40 mL, condition (b): 800 mL) to wash away impurities from the anion exchange chromatography column. After that, the (FpL_C3KX 9a)5-IT-3K adsorbed on SuperQ was eluted using Buffer D (condition (a): 30 mL, condition (b): 600 mL) containing 300 mmol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name), and fractions containing the polypeptide were collected.

[0147] (2-4) A washing step was carried out to wash away proteins remaining in the SuperQ column using buffer D (condition (a): 20 mL, condition (b): 400 mL) containing 1 mol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name).

[0148] (2-5) The purity of (FpL_C3KX 9a)5-IT-3K contained in the fractions collected in (1-3) and (2-4) was measured and the amount of the polypeptide recovered was calculated using the same method as in Example 4(4). Note that under condition (b), the purity of (FpL_C3KX 9a)5-IT-3K contained in the two fractions was also confirmed by SDS-PAGE.

[0149] The chromatograms obtained under each condition in this example are shown in Figure 9. Under all conditions and at all purification stages, a peak corresponding to the FpL polypeptide (white arrow in Figure 9) was confirmed.

[0150] The SDS-PAGE results of the fractions obtained at each purification step under condition (b) are shown in Figure 10. In fraction (A) obtained in the second purification step, only the band corresponding to (FpL_C3KX 9a)5-IT-3K (white arrow in Figure 10) was observed, suggesting that the polypeptide was highly purified.

[0151] The purification results under each condition in this example are shown in Table 14. The purity of the FpL polypeptide (referred to as "FpL" in Table 14) contained in the elution fraction after the second purification step was 90% or more under all conditions, demonstrating that the polypeptide can be purified to a high purity even if the order of chromatography is reversed. It can be seen that...

[0152] [Table 14]

[0153] Example 10: Purification using a combination of an anion exchange chromatography column and a hydrophobic chromatography column (part 4) In Example 9, purification was performed using a sample containing (FpL_C3KX 9a)5-IT-3K (sequence number 7) from the purification targets obtained in Example 1. However, we investigated whether the effect of improving purity could also be achieved when using a sample containing (FpL_C3KX 7e)4-IT-3K (sequence number 5) from the purification targets obtained in Example 1.

[0154] (1) First-stage purification (purification using a hydrophobic chromatography column) (1-1) Ammonium sulfate and sodium chloride were added to a sample containing (FpL_C3KX 7e)4-IT-3K (SEQ ID NO: 5) from the purification target obtained in Example 1, and the mixture was stirred at room temperature for 3 hours, after which the supernatant was collected by centrifugation. Table 15 summarizes the sample volume, the amount of ammonium sulfate and sodium chloride added, and the protein concentration in the supernatant measured by the method described in Example 2(5) under each of the conditions (conditions (a) and (b)) examined in this example.

[0155] [Table 15]

[0156] (1-2) The supernatant prepared in (1-1) was applied to a phenyl-packed column (phenyl column) pre-equilibrated with buffer A containing ammonium sulfate and sodium chloride, allowing (FpL_C3KX 7e)4-IT-3K to be adsorbed onto the phenyl. The volume of the phenyl column and the concentrations of ammonium sulfate and sodium chloride contained in the equilibration solution under each condition are shown in Table 15. The load amount onto the phenyl column is also shown in Table 15.

[0157] (1-3) A pre-elution wash step was performed to wash away impurities from the phenyl column using the buffer solution used to equilibrate the phenyl column (condition (a): 60 mL, condition (b): 2400 mL). Then, the (FpL_C3KX 7e)4-IT-3K adsorbed to the phenyl was eluted using buffer solution A (condition (a): 30 mL, condition (b): 1200 mL), and fractions containing the polypeptide were collected.

[0158] (2) Second-stage purification (purification using an anion exchange chromatography column) (2-1) The fractions collected in (1-3) were diluted two-fold with buffer solution D containing 0.025% (w / v) Tween 20 (trade name, manufactured by Tokyo Chemical Industry Co., Ltd.).

[0159] (2-2) The sample diluted in (2-1) was applied to a column packed with SuperQ (SuperQ column) pre-equilibrated with buffer D containing 200 mmol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name), allowing (FpL_C3KX 7e)4-IT-3K to be adsorbed onto the SuperQ. The SuperQ column capacity, sodium chloride concentration in the equilibration solution, and protein concentration in the sample measured by the method described in Example 2(5) under each condition are shown in Table 16. The load amount onto the SuperQ column is also shown in Table 16.

[0160] [Table 16]

[0161] (2-3) A pre-elution wash step was performed using Buffer D (condition (a): 40 mL, condition (b): 1600 mL) to wash away impurities from the anion exchange chromatography column. After that, the (FpL_C3KX 7e)4-IT-3K adsorbed on SuperQ was eluted using Buffer D (condition (a): 30 mL, condition (b): 1200 mL) containing 300 mmol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name), and fractions containing the polypeptide were collected. (2-4) A washing step was carried out to wash away proteins remaining in the SuperQ column using buffer D (condition (a): 20 mL, condition (b): 800 mL) containing 1 mol / L sodium chloride and 0.025% (w / v) Tween 20 (trade name).

[0162] (2-5) The purity of (FpL_C3KX 7e)4-IT-3K contained in the fractions collected in (1-3) and (2-4) was measured and the recovered amount of the polypeptide was calculated using the same method as in Example 4(4). Note that under condition (b), the purity of (FpL_C3KX 7e)4-IT-3K contained in the two fractions was also confirmed by SDS-PAGE.

[0163] The chromatograms obtained under each condition in this example are shown in Figure 11. Under all conditions and at all purification stages, a peak corresponding to the FpL polypeptide (white arrow in Figure 11) was confirmed.

[0164] The SDS-PAGE results of the fractions obtained at each purification step under condition (b) are shown in Figure 12. In fraction (A) obtained in the second purification step, only the band corresponding to (FpL_C3KX 7e)4-IT-3K (white arrow in Figure 12) was confirmed, suggesting that the polypeptide was highly purified.

[0165] The purification results under each condition in this example are shown in Table 17. The purity of the FpL polypeptide (referred to as "FpL" in Table 17) contained in the elution fraction after the second purification step was 90% or higher under all conditions, demonstrating that the polypeptide can be purified to a high purity even when a sample containing (FpL_C3KX 7e)4-IT-3K (SEQ ID NO: 5), one of the purification targets obtained in Example 1, is used.

[0166] [Table 17]

[0167] Example 11 Purification using a combination of an anion exchange chromatography column and a hydrophobic chromatography column (part 5) (1) First-stage purification (purification using a hydrophobic chromatography column) (1-1) To a 29 mL sample containing (FpL_C3KX 9a)5-IT-3K (SEQ ID NO: 7) from the purification target obtained in Example 1, 0.9 mol / L ammonium sulfate and 0.6 mol / L sodium chloride were added, and the mixture was stirred at room temperature for 3 hours. 50 mL of the supernatant was recovered by centrifugation. Under the conditions examined in this example, the protein concentration in the supernatant, as measured by the method described in Example 2(5), was 6.1 mg / mL.

[0168] (1-2) The supernatant prepared in (1-1) was applied to a 1 mL phenyl column pre-equilibrated with buffer A containing 0.9 mol / L ammonium sulfate and 0.6 mol / L sodium chloride, and (FpL_C3KX 9a)5-IT-3K was adsorbed onto the phenyl. The loading amount of the phenyl column was 305 mg / mL (carrier).

[0169] (1-3) After a pre-elution wash step in which impurities in the phenyl column were washed away using 12 mL of the buffer solution used to equilibrate the phenyl column, (FpL_C3KX 9a)5-IT-3K adsorbed to the phenyl column was eluted using 6 mL of buffer solution A, and fractions containing the polypeptide were collected.

[0170] (2) Second-stage purification (purification using an anion exchange chromatography column) The fractions collected in (2-1) and (1-3) were diluted 10-fold with buffer A, buffer D containing 0.1% (w / v) Tween 20 (trade name, manufactured by Tokyo Chemical Industry Co., Ltd.), or buffer E (50 mmol / L sodium phosphate buffer (pH 6.0)) containing 0.1% (w / v) Tween 20 (trade name). Table 18 summarizes the sample volume used, the pH of the diluted buffer, and the Tween 20 (trade name) concentration contained in the diluted buffer under each of the conditions (conditions (a), (b), and (c)) examined in this example.

[0171] [Table 18]

[0172] (2-2) 9 mL of the sample diluted in (2-1) was applied to a column packed with 1 mL of SuperQ (SuperQ column) that had been pre-equilibrated with a buffer containing sodium chloride and Tween 20 (trade name), allowing (FpL_C3KX 9a)5-IT-3K to be adsorbed onto the SuperQ. The pH of the equilibration solution, the sodium chloride concentration and Tween 20 (trade name) concentration contained in the equilibration solution, and the protein concentration of the sample measured by the method described in Example 2(5) under each condition are shown in Table 18. The load amount onto the SuperQ column is also shown in Table 18.

[0173] (2-3) A pre-elution wash step was performed using 20 mL of the same buffer solution used to equilibrate the SuperQ column to wash away impurities from the anion exchange chromatography column. The (FpL_C3KX 9a)5-IT-3K adsorbed to the SuperQ column was then eluted with the buffer solution prepared by diluting the sample in (2-1) containing 300 mmol / L sodium chloride and Tween 20 (trade name), and fractions containing the polypeptide were collected. The pH and Tween 20 (trade name) concentrations of the eluates under each condition are shown in Table 18.

[0174] (2-4) A washing step was carried out to wash away proteins remaining in the SuperQ column using the buffer prepared by diluting the sample in (2-1) and containing 1 mol / L sodium chloride and Tween 20 (trade name). The pH of the washing solution and the concentration of Tween 20 (trade name) contained in the washing solution under each condition are as shown in Table 18.

[0175] (2-5) Using a method similar to that described in Example 4(4), the purity of (FpL_C3KX 9a)5-IT-3K contained in the fractions recovered in (1-3) and (2-4) was measured, and the recovered amount of the polypeptide was calculated.

[0176] The chromatograms obtained under each condition in this example are shown in Figure 13. Under all conditions and at all purification stages, a peak corresponding to the FpL polypeptide (white arrow in Figure 13) was confirmed.

[0177] The purification results under each condition in this example are shown in Table 19. The purity of the FpL polypeptide (referred to as "FpL" in Table 19) contained in the elution fraction after the second purification step was 90% or higher under all conditions.

[0178] [Table 19] < / ii> < / ii>

Claims

1. Culturing a recombinant Escherichia coli strain containing a polynucleotide encoding a polypeptide containing at least the immunoglobulin-binding domain of Protein L (FpL) derived from a bacterium of the genus Finegoldia to express the polypeptide; and purifying the polypeptide from the culture of the recombinant E. coli obtained in the expression step by using chromatography, the step of purifying the polypeptide comprises a purification step using anion exchange chromatography and a purification step using hydrophobic chromatography; The purification step using hydrophobic chromatography includes: equilibrating a hydrophobic chromatography column, applying a sample containing the polypeptide to the column, and recovering the polypeptide; At least one of the solution for equilibrating the column and the sample contains ammonium sulfate at a concentration of 0.4 mol / L or more and 1.1 mol / L or less. method.

2. 10. The method of claim 1, wherein at least one of the solution for equilibrating the column and the sample further comprises sodium chloride.

3. The production method according to claim 1 or 2, wherein the polypeptide comprising at least the immunoglobulin-binding domain of FpL is a polypeptide selected from the group consisting of: (a) a polypeptide comprising at least amino acid residues consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide having an amino acid sequence comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having immunoglobulin-binding activity; (c) A polypeptide comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, having an identity of 70% or more to the amino acid sequence consisting of said amino acid residues, and having immunoglobulin-binding activity.

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