Cell separation solvent composition

Abstract

To provide a cell separation solvent that allows a large number of cells to be separated and refined in a short time under animal-free conditions in the preparation of cells for regenerative therapy, and a composition thereof.SOLUTION: Instead of conventionally used solvent compositions for cell separation comprising animal-derived components such as BSA and FBS, a solvent composition comprising a salt of an organic acid and a chelator is used.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a solvent composition used to separate and purify cells by suspending at least one type of cell and contacting the cell with an adsorbent, and particularly to a solvent composition that does not contain animal-derived components. [Background technology]

[0002] BC2LCN lectin, derived from the N-terminal domain of BC2L-C lectin produced by the Gram-negative bacterium Burkholderia cenocepacia, is a protein with binding affinity to glycans containing fucose residues. For example, it is known to have high binding affinity to several types of glycans containing fucose residues, such as H-type 1 glycans (Fucα1-2Galβ1-3GlcNAc) and H-type 3 glycans (Fucα1-2Galβ1-3GalNAc), which are known as undifferentiated glycan markers described in Non-Patent Document 1, Patent Document 1, and Patent Document 2, as well as Lewis Y glycans (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and Lewis X glycans (Galβ1-4(Fucα1-3)GlcNAc) (Non-Patent Document 2).

[0003] Furthermore, BC2LCN lectin is known to bind to undifferentiated human iPS cells and human ES cells in which H-type 1 glycans and H-type 3 glycans are highly expressed, but not to human somatic cells (Non-Patent Document 3). Furthermore, because BC2LCN lectin has the ability to bind to the undifferentiated glycan markers, it has been used, for example, to detect glycoconjugates containing undifferentiated glycan markers and to detect undifferentiated cells such as human iPS cells and human ES cells (Patent Documents 1 and 2). Furthermore, H-type 1 glycans are known to be highly expressed in certain cancer cells as SSEA-5 (Non-Patent Document 4).

[0004] Although BC2LCN lectin has the same ability to detect undifferentiated stem cells as anti-Nanog antibodies, which are known antibodies for detecting undifferentiated cells (Patent Document 2), the binding between BC2LCN lectin and the glycans of undifferentiated cells is due to electrostatic interaction, and the strength of the binding is affected by the external environment, such as the solvent and salt concentration. Therefore, depending on the experimental conditions, it is possible that the binding affinity between the glycans and BC2LCN lectin may be reduced when detecting the undifferentiated cells and / or glycoconjugates containing the undifferentiation glycan marker. Therefore, there is a demand for a BC2LCN lectin with improved binding affinity to the undifferentiation glycan marker.

[0005] One known method for improving protein function is to introduce amino acid mutations into proteins using protein engineering techniques to improve the target function. For example, an Fc-binding protein is known in which specific amino acid residues have been replaced with other amino acid residues to improve its stability against heat, acid, and / or alkali (Patent Document 3). Patent Document 4 also discloses a BC2LCN lectin that has been produced using a similar technique, with improved heat stability and binding affinity to sugar chains due to amino acid substitutions at specific positions (Patent Document 4). Furthermore, a BC2LCN lectin with improved productivity when produced using a microorganism such as Escherichia coli is known (Patent Document 5).

[0006] Furthermore, cell separation and purification methods that utilize an adsorbent in which BC2LCN lectin, which has improved functions such as heat stability and binding affinity to glycans and productivity through microbial expression, is immobilized on an insoluble carrier include a method for removing undifferentiated cells such as iPS cells and separating and purifying differentiated cells (Patent Document 6), and a method for detaching and recovering undifferentiated cells adsorbed to an adsorbent (Patent Document 7).

[0007] However, while other cell separation and purification methods, such as those using magnetic beads (Non-Patent Document 5), cell sorting using a cell sorter (Non-Patent Document 5), and cell rolling columns (Non-Patent Document 6), have been known, it has been extremely difficult to prepare high-quality, pure iPS cells for clinical use using these methods. For example, iPS cell culture has traditionally been performed using culture conditions that use fetal bovine serum (FBS) or mouse-derived feeder cells, which are an FBS substitute. However, serum and feeder cells contain unknown factors, which can vary between lots, and they may contain viruses or unknown pathogens. Therefore, to reduce the risk of pathogen contamination and to obtain highly reproducible research results, a medium with a known composition using as purified or synthetic components as possible has been desired.

[0008] Furthermore, the cell separation solvent used in the cell separation process has generally been, for example, MACS buffer (phosphate buffer solution (D-PBS(-)) and 2 mM ethylenediaminetetraacetic acid (EDTA) to which 0.5% (w / v) bovine serum albumin (BSA), an animal-derived component, has been added), or a method containing the addition of fetal bovine serum (FBS) or human serum albumin (HSA) to adjust the osmotic pressure of the cells and prevent cell death. However, as with the cell culture process, there have been concerns about a decline in cell quality due to the incorporation of animal-derived components into iPS cells. Therefore, in order to prepare iPS cells for clinical use in regenerative medicine, efforts are currently underway worldwide to develop culture media with compositions that do not contain animal-derived components and that allow for culture under animal-free conditions.In addition, methods have been developed for using TrypLE Select Enzyme (recombinant trypsin), which does not contain animal-derived components, as a stripping solvent for iPS cell preparation, and for using a cell suspension solvent containing recombinant HSA during cell separation and purification.However, both of these reagents are expensive and have the problem of not being able to be used to prepare large quantities of cells.

[0009] However, at present, there is a strong demand for the development of a solvent composition for separating and purifying cells that does not contain animal-derived components, which would enable the inexpensive separation and purification of large quantities of cells under animal-free conditions in order to prepare iPS cells for regenerative medicine applications. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2013 / 065302 issue [Patent Document 2] WO2013 / 128914 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-206046 [Patent Document 4] Patent Publication No. 2020-025535 [Patent Document 5] Patent Publication No. 2020-058343 [Patent Document 6] Patent Publication No. 2018-134073 [Patent Document 7] Patent Publication No. 2019-000063 [Non-patent literature]

[0011] [Non-Patent Document 1] Tateno, H et al., Stem Cells Transl Med.2013, 2(4):265-273. [Non-patent document 2] Sulak, O et al., Structure.2010, 18(1):59-72. [Non-patent document 3] Tateno, H et al., J Biol Chem. 2011, 286(23):20345-20353. [Non-patent document 4] Tang, C et al., Nat Biotechnol. 2011, 29(9):829-835. [Non-patent document 5] Fong CY et al., Stem Cell Rev Rep.2009, 5(1):72-80. [Non-patent document 6] Mahara, A et al., J Biomater Sci Polym Ed.2014, 25(14-15):1590-601. Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a solvent composition free of animal-derived components for separating and purifying a large amount of cells in a short time under animal-free conditions in the preparation of cells for regenerative medicine. Specifically, the object of the present invention is to provide a solvent composition free of animal-derived components for simply and efficiently separating undifferentiated cells that express undifferentiation markers on their cell surface, such as sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, in a method for separating undifferentiated cells, which comprises the steps of contacting a cell mixture with an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier and then detaching the cells adsorbed to the adsorbent. [Means for solving the problem]

[0013] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that by using a solvent composition containing an organic acid salt and a chelating agent instead of the conventionally used solvent composition for cell separation containing animal-derived components such as BSA and FBS, it is possible to separate and purify a large amount of cells in a short period of time under animal-free conditions, and thus completed the present invention.

[0014] That is, the present invention includes the inventions described in [1] to

[13] below. [1] A solvent composition used to separate and purify cells by suspending at least one type of cell and contacting it with an adsorbent, characterized in that the solvent composition contains a salt of an organic acid and a chelating agent in an aqueous solvent and does not contain animal-derived components. [2] The solvent composition according to [1] above, wherein the organic acid is one selected from acetic acid, citric acid, and gluconic acid. [3] The solvent composition according to [1] or [2] above, wherein the salt of the organic acid is a sodium salt or a potassium salt. [4] The solvent composition according to any one of [1] to [3] above, wherein the concentration of the organic acid is 0.1 mol / L or more and 1.0 mol / L or less. [5] The solvent composition according to any one of [1] to [4] above, wherein the chelating agent is either ethylenediaminetetraacetic acid or ethylene glycol bis(2-aminoethyl ether)tetraacetic acid. [6] The solvent composition according to any one of [1] to [5] above, wherein the adsorbent is an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier, and the solvent composition is used to separate and purify cells by adsorbing the cells onto the adsorbent and then desorbing the cells from the adsorbent. [7] A method for separating and purifying cells, comprising: adsorbing cells onto an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier using the solvent composition according to any one of [1] to [6] above; and then desorbing the cells from the adsorbent using the solvent composition according to any one of claims 1 to 6, which contains fucose. [8] A method for separating and purifying cells according to [7] above, wherein the cells have glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or glycans containing a structure consisting of Fucα1-2Galβ1-3GalNAc. [9] A method for separating and purifying cells according to [8] above, wherein the cells having glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or glycans containing a structure consisting of Fucα1-2Galβ1-3GalNAc are human iPS cells.

[0015]

[10] A method for separating and purifying the cells according to any one of [7] to [9] above, wherein the fucose-binding protein is any one of the following (a) to (d): (a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or greater. (b) A fucose-binding protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and which has binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, where X is an integer of 120 or more. (c) A fucose-binding protein comprising an amino acid sequence containing one or more of the amino acid substitutions listed in (1) to (3) below in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or greater: (1) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (2) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (3) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (d) A fucose-binding protein comprising the amino acid sequence of the fucose-binding protein of (c) above, in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1, and having binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0016]

[11] A method for separating and purifying the cells according to any one of [7] to [9] above, wherein the fucose-binding protein is any one of the following (e) to (h): (e) A fucose-binding protein consisting of an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is further added at the N-terminus and an oligopeptide containing cysteine ​​is further added at the C-terminus, wherein X is an integer of 120 or greater. (f) A fucose-binding protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and further comprising a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, wherein the fucose-binding protein has binding affinity to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, and X is an integer of 120 or more. (g) A fucose-binding protein comprising an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing cysteine ​​is added at the C-terminus, and which comprises one or more of the amino acid substitutions listed in (4) to (6) below, wherein X is an integer of 120 or greater: (4) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (6) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) A fucose-binding protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of (g) above, and further having an oligopeptide containing a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, and having binding affinity for glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0017]

[12] A method for separating and purifying cells according to any one of [7] to [9], characterized in that a hydrophilic polymer is covalently immobilized on a water-insoluble carrier.

[13] A method for separating and purifying cells according to any one of [7] to [9] above, characterized by using an adsorbent packed in a column.

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

[0019] The solvent composition of the present invention (hereinafter sometimes abbreviated as the composition of the present invention) is a solvent composition used to separate and purify cells by suspending at least one type of cell and contacting it with an adsorbent, and is characterized by not containing animal-derived components. The composition of the present invention is particularly suitable for use in separating and purifying cells by adsorbing cells to an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier and then detaching the cells from the adsorbent.

[0020] Since the composition of the present invention does not contain animal-derived components, it not only reduces the risk of contamination with viruses or unknown pathogens, but also prevents deterioration in quality due to the uptake of animal-derived components into cells.

[0021] The composition of the present invention is characterized by containing a salt of an organic acid and a chelating agent in a water-soluble solvent. The organic acid in the composition of the present invention is not particularly limited as long as it is water-soluble and does not exhibit cytotoxicity, and examples thereof include monocarboxylic acids such as acetic acid, propionic acid, and butyric acid, dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, and adipic acid, tricarboxylic acids such as citric acid, and sugar-derived carboxylic acids such as glucuronic acid and gluconic acid. Among these, acetic acid, citric acid, and gluconic acid are preferred because they have low cytotoxicity and allow cells to be separated and purified with high efficiency in the cell separation and purification method described below.

[0022] Furthermore, the salt of the organic acid is preferably a sodium salt or a potassium salt, since they are highly water-soluble and do not adversely affect cells. The concentration of the organic acid is not particularly limited as long as it does not kill cells, but by setting the concentration to 0.1 mol / L or more and 1.0 mol / L or less, damage to cells due to cell shrinkage or cell hypertrophy can be suppressed.

[0023] Furthermore, in addition to the organic acid salts described above, the composition of the present invention may contain a compound that promotes cell detachment from the adsorbent in the cell separation and purification method described below. Specifically, by containing a chelating agent, the target cells can be detached from the adsorbent with high efficiency. The chelating agent contained in the composition of the present invention is not particularly limited as long as it is water-soluble and does not exhibit cytotoxicity, but ethylenediaminetetraacetic acid or ethylene glycol bis(2-aminoethyl ether)tetraacetic acid is preferred in terms of its ability to detach the target cells from the adsorbent with high efficiency.

[0024] As described above, the composition of the present invention is suitable for use in separating and purifying cells by adsorbing cells to an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier and then detaching the cells from the adsorbent. Therefore, in addition to the organic acid salts and chelating agents described above, the composition of the present invention may contain fucose, a compound that dissociates the bond between cells and the fucose-binding protein. The composition of the present invention may also contain a compound that regulates the osmotic pressure of cells, a specific example of which is mannitol. Furthermore, in addition to the organic acid salts, chelating agents, compounds that dissociate the bond between cells and the fucose-binding protein, and compounds that regulate the osmotic pressure of cells, the composition of the present invention may also contain compounds that are water-soluble, non-cytotoxic, and not derived from animals, depending on the type of cells and the separation and purification method.

[0025] Next, the method for separating and purifying cells of the present invention (hereinafter sometimes abbreviated as the separation and purification method of the present invention) will be described. The separation and purification method of the present invention is a method for separating and purifying cells by using an adsorbent in which a fucose-binding protein is immobilized on an insoluble carrier and the composition of the present invention described above, to adsorb cells to the adsorbent, and then detaching the cells from the adsorbent.

[0026] Specifically, one example of a method involves contacting at least one type of cell with an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier, and then obtaining cells that did not adsorb to the adsorbent and cells detached from the adsorbent by adding a fucose-containing composition of the present invention. Furthermore, cells that bind weaker to the fucose-binding protein are detached from the adsorbent at lower concentrations of fucose, whereas cells that bind stronger to the fucose-binding protein are detached from the adsorbent at higher concentrations of fucose. Therefore, by sequentially adding compositions of the present invention containing different fucose concentrations, cells with different binding strengths to the fucose-binding protein can be detached from the adsorbent and obtained. The fucose concentration in the fucose-containing composition of the present invention used to detach cells adsorbed to the adsorbent is not particularly limited as long as it does not kill the cells, but is preferably 0.001 mol / L or more and 1.0 mol / L or less, and more preferably 0.01 mol / L or more and 0.5 mol / L or less.

[0027] The separation and purification method of the present invention uses an adsorbent in which a fucose-binding protein (described later) is immobilized on an insoluble carrier. This allows cells having fucose-containing glycans to which the fucose-binding protein binds, specifically, H-type 1 glycans (structures consisting of Fucα1-2Galβ1-3GlcNAc), H-type 3 glycans (structures consisting of Fucα1-2Galβ1-3GalNAc), Lewis Y glycans (structures consisting of Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), and Lewis b glycans (structures consisting of Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), to be separated and purified, and cells not having these glycans can be obtained. Specific examples of cells having the aforementioned glycans include undifferentiated cells and cancer cells. Examples of undifferentiated cells include human iPS cells and human ES cells. Examples of cancer cells include human embryonal carcinoma cells such as 2102Ep and NT2 / D1, human lung adenocarcinoma cells such as PC-9, human pancreatic cancer cells such as Capan-1, and human colon cancer cells such as HT29. Any of these cells may have sugar chains containing a structure consisting of, for example, Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0028] Next, the fucose-binding protein in the separation and purification method of the present invention will be described. The fucose-binding protein in the separation and purification method of the present invention is a protein that has binding properties to fucose-containing sugar chains such as H-type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc), H-type 3 sugar chain (Fucα1-2Galβ1-3GalNAc), Lewis Y sugar chain (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), and Lewis b sugar chain (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), and the aforementioned BC2LCN lectin is also included in the fucose-binding protein in the separation and purification method of the present invention. Specifically, the fucose-binding protein in the separation and purification method of the present invention is a protein expressed as a recombinant protein in an Escherichia coli transformant: (a) a protein comprising the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence of recombinant BC2LCN lectin shown in SEQ ID NO: 1 (which corresponds to the amino acid sequence from the second to the 156th amino acid in the amino acid sequence registered with GenPept under registration number WP_006490828), where X is an integer of 120 or greater; or (b) a protein consisting of an amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been deleted, substituted, or added, and which has the ability to bind to H-type 1 glycans and / or H-type 3 glycans, where X is an integer of 120 or greater. As long as the fucose-binding protein in the separation and purification method of the present invention has the ability to bind to the fucose-containing sugar chain, particularly a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acids may be deleted, substituted, or added in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1, for example, 15 or fewer, preferably 10 or fewer amino acids may be deleted, substituted, or added.

[0029] Furthermore, X may be 120 or more and 155 or less, or 125 or more and 155 or less. As disclosed in JP 2020-25535 A, the fucose-binding protein in the separation and purification method of the present invention can have improved productivity (expression amount) by deleting multiple amino acid residues on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1, compared to when the amino acid residues are not deleted.

[0030] Furthermore, in order to improve heat stability, the fucose-binding protein in the separation and purification method of the present invention may contain one or more of the following: (i) substitution of the glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue; (ii) substitution of the cysteine ​​residue at position 72 in the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and / or an alanine residue; and (iii) substitution of the glutamine residue at position 65 in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue.

[0031] As disclosed in JP 2020-25535 A, the heat stability of the fucose-binding protein in the separation and purification method of the present invention can be improved by making the amino acid substitutions described in (i) to (iii) above. The amino acid substitutions described in (i) to (iii) above are effective in improving heat stability whether used alone or in combination, but combining multiple amino acid substitutions described in (i) to (iii) above is more preferable in terms of further improving heat stability.

[0032] Furthermore, as long as the fucose-binding protein in the separation and purification method of the present invention has the ability to bind to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acid residues may be deleted, substituted, or inserted in a region other than the positions substituted by the substitutions (1) to (3) in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1; for example, 15 or fewer, preferably 10 or fewer amino acid residues may be deleted, substituted, or inserted.

[0033] The fucose-binding protein in the cell separation of the present invention may have an additional amino acid sequence at its N-terminus and / or C-terminus that is useful for detecting the fucose-binding protein, as long as it has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

[0034] Examples of the additional amino acid sequence include an oligopeptide containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose-binding protein, cellulose-binding domain, myc tag, FLAG tag, etc. Among these additional amino acid sequences, an oligopeptide containing a polyhistidine sequence or GST is preferred, and an oligopeptide containing a polyhistidine sequence is more preferred, because they have high productivity when produced using Escherichia coli and the fucose-binding protein can be easily detected using a fluorescently labeled anti-polyhistidine antibody or anti-GST antibody.

[0035] The number of histidine repeats in an oligopeptide containing a polyhistidine sequence is not particularly limited, but a short histidine repeat sequence makes detection with an anti-polyhistidine antibody difficult, while a long histidine repeat sequence may impair the binding ability of the fucose-binding protein to the sugar chain. Therefore, the length of the histidine repeat sequence in an oligopeptide containing a polyhistidine sequence is preferably a repeat sequence consisting of 5 to 15 histidines, and more preferably a repeat sequence consisting of 5 to 10 histidines. The position at which the oligopeptide containing a polyhistidine sequence is added to the fucose-binding protein is not particularly limited, and may be both the N-terminus and the C-terminus, or either the N-terminus or the C-terminus. However, in terms of efficient detection with an anti-polyhistidine antibody, the oligopeptide containing a polyhistidine sequence is preferably added to the N-terminus of the fucose-binding protein.

[0036] Furthermore, the fucose-binding protein in the separation and purification method of the present invention may have an additional amino acid sequence at its N-terminus and / or C-terminus consisting of an oligopeptide containing a cysteine ​​residue or a lysine residue (hereinafter referred to as a tag for carrier immobilization), which is useful for immobilizing the fucose-binding protein on a water-insoluble carrier. By immobilizing the fucose-binding protein on a carrier, it is possible to prepare an adsorbent for undifferentiated cells, such as human iPS cells, as described in Patent Document 4, for example.

[0037] The length of the tag for immobilization on a carrier is not particularly limited, as long as the fucose-binding protein has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. As the tag for immobilization on a carrier, an oligopeptide consisting of 2 to 10 amino acid residues and containing one or more cysteine ​​residues is preferred, as it allows highly selective and efficient immobilization to a water-insoluble carrier. Specific examples of the tag for immobilization on a carrier include an oligopeptide consisting of the three amino acid residues "Gly-Gly-Cys," an oligopeptide consisting of the five amino acid residues "Ala-Ser-Gly-Gly-Cys," and an oligopeptide consisting of the seven amino acid residues "Gly-Gly-Gly-Ser-Gly-Gly-Cys." There are no particular limitations on the position at which the oligopeptide containing one or more cysteines is added to the fucose-binding protein, and it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus. However, it is preferable that the oligopeptide containing one or more cysteine ​​residues is added to the C-terminus of the fucose-binding protein, because this allows for efficient immobilization of the fucose-binding protein to the carrier and, further, because it is away from the active center of the fucose-binding protein and therefore is less likely to inhibit the binding activity.

[0038] Specific examples of fucose-binding proteins in the separation and purification method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (the amino acid sequence from position 1 to position 127 of the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 (the amino acid sequence in which the 72nd cysteine ​​residue of SEQ ID NO: 2 is substituted with a glycine residue), SEQ ID NO: 4 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), SEQ ID NO: 5 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue, the 65th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), SEQ ID NO: 6 (the amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus ), SEQ ID NO: 7 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 8 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 9 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 4 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), and SEQ ID NO: 10 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 5 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus).

[0039] In the separation and purification method of the present invention, a signal peptide may be added to the N-terminus of the fucose-binding protein to promote efficient expression in the host. When the host is Escherichia coli, examples of the signal peptide include signal peptides that direct protein secretion into the periplasm, such as PelB, DsbA, MalE, and TorT.

[0040] The DNA encoding the fucose-binding protein in the separation and purification method of the present invention can be prepared by known methods. Examples of methods for preparing the DNA include a method in which the amino acid sequence of the fucose-binding protein in the separation and purification method of the present invention is converted to a nucleotide sequence and DNA containing the nucleotide sequence is artificially synthesized, a method in which DNA encoding the fucose-binding protein in the separation and purification method of the present invention is directly artificially prepared, and a method in which DNA is prepared from the genomic DNA of Burkholderia cenocepacia using a DNA amplification method such as PCR.

[0041] In this preparation method, when designing the base sequence, it is preferable to consider the codon usage frequency in the E. coli to be transformed. For example, AGA, AGG, CGG, or CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are all rarely used codons (rare codons), so it is preferable to select and convert codons other than these codons. Analysis of codon usage frequency can also be done using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website, http: / / www.kazusa.or.jp / codon / , accessed May 7, 2020).

[0042] To transform Escherichia coli with DNA encoding a fucose-binding protein prepared by the above-mentioned method, the DNA itself may be used for transformation. However, it is preferable to use an expression vector prepared by inserting the DNA into an appropriate position in a vector based on a bacteriophage, cosmid, or plasmid, which is commonly used for transforming prokaryotic and eukaryotic cells, in order to achieve stable transformation. Here, "appropriate position" means a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, and regions involved in transferability. Furthermore, when inserting the DNA into a vector, it is preferably inserted into the vector in a state where it is linked to functional DNA, such as a promoter, required for expression.

[0043] The vector used as the expression vector is not particularly limited as long as it can stably exist and replicate in the host, and examples thereof include pET vectors, pUC vectors, pTrc vectors, pCDF vectors, and pBBR vectors. Examples of the promoter include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter, as well as the λPL promoter and λPR promoter of λ phage. Transformation of the host Escherichia coli using the expression vector can be carried out using a method commonly used by those skilled in the art. For example, when selecting the host strain Escherichia coli JM109, Escherichia coli BL21(DE3), Escherichia coli NiCo21(DE3), or Escherichia coli W3110, methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, vol. 256, 1992) can be used.

[0044] Next, a method for producing the fucose-binding protein of the present invention will be described. The fucose-binding protein of the present invention can be produced by a process comprising two steps: a step of producing the fucose-binding protein by culturing the transformant (hereinafter referred to as step 1), and a step of recovering the fucose-binding protein from the resulting culture (hereinafter referred to as step 2). In this specification, the culture includes not only the cultured transformant cells themselves and cell secretions, but also the medium used for the culture. In step 1, the transformant may be cultured in a medium suitable for its culture. For example, when Escherichia coli is used as the host, it is preferable to use Terrific Broth (TB) medium, Luria-Bertani (LB) medium, or the like supplemented with the necessary nutrients.

[0045] When the expression vector contains a drug resistance gene, selective growth of the transformant can be achieved by adding a drug corresponding to the gene to the medium and performing the first step. For example, when the expression vector contains a kanamycin resistance gene, it is preferable to add kanamycin to the medium. The culture temperature may be within a range generally known for the host used. For example, when the host is Escherichia coli, it is 10°C to 40°C, preferably 20°C to 37°C, and may be appropriately determined taking into consideration the production yield of the fucose-binding protein of the present invention, etc. The pH of the medium may be within a range generally known for the host used. For example, when the host is Escherichia coli, it is within a range of pH 6.8 to pH 7.4, preferably around pH 7.0, and may be appropriately determined taking into consideration the production yield of the fucose-binding protein of the present invention, etc. When an inducible promoter is introduced into the expression vector, expression of the fucose-binding protein of the present invention can be induced by adding an inducer to the medium under conditions that allow good production of the protein.

[0046] A preferred inducer is isopropyl-β-D-thiogalactopyranoside (IPTG), for example, when a tac promoter or lac promoter is used, and its concentration ranges from 0.005 mM to 1.0 mM, preferably from 0.01 mM to 0.5 mM. Expression induction by addition of IPTG may be carried out under conditions generally known for the host used. In the second step, the fucose-binding protein of the present invention is recovered from the culture obtained in the first step by a generally known recovery method. For example, when the fucose-binding protein of the present invention is secreted and produced in the culture medium, the cells can be separated by centrifugation, and the fucose-binding protein of the present invention can be recovered from the resulting culture supernatant. When the fucose-binding protein of the present invention is expressed intracellularly (including the periplasm in prokaryotes), the cells can be collected by centrifugation and then disrupted by adding an enzyme treatment agent, surfactant, or the like, and the fucose-binding protein can be recovered from the cell lysate. Furthermore, when it is desired to improve the purity of the fucose-binding protein, methods known in the art may be used, such as a separation and purification method using liquid chromatography.

[0047] As the liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. are preferably used, and a combination of these chromatographies is more preferable. The purity and molecular weight of the fucose-binding protein of the present invention purified by the above chromatography can be determined by methods known in the art, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography.

[0048] The binding affinity of the fucose-binding protein of the present invention to sugar chains can be evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, or the like. Surface plasmon resonance will be described as an example. Binding affinity evaluation by surface plasmon resonance can be performed, for example, using a Biacore T200 instrument (GE Healthcare) with the fucose-binding protein as the analyte and sugar chains (sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc) as the solid phase. Sensor chips with immobilized sugar chains can be prepared using biotin-labeled sugar chains, either on a streptavidin-coated sensor chip (Sensor Chip SA, GE Healthcare) or on a dextran-coated sensor chip (Sensor Chip CM5, GE Healthcare) on which streptavidin has been immobilized. Furthermore, binding affinity evaluation can be performed using the kinetic analysis program provided with the instrument.

[0049] Next, the adsorbent in the separation and purification method of the present invention will be described. There are no particular limitations on the raw material of the water-insoluble carrier used for the adsorbent in the separation and purification method of the present invention, and examples thereof include inorganic carriers such as silica gel and glass on which a thin gold film has been vapor-deposited; water-insoluble polysaccharide carriers made from polysaccharides such as agarose, cellulose, chitin, and chitosan, and crosslinked polysaccharide carriers obtained by crosslinking these with a crosslinking agent; crosslinked polysaccharide carriers obtained by crosslinking water-soluble polysaccharides such as dextran, pullulan, starch, alginate, and carrageenan with a crosslinking agent; synthetic polymer carriers such as poly(meth)acrylate, polyvinyl alcohol, polyurethane, and polystyrene, and crosslinked synthetic polymer carriers obtained by crosslinking these with a crosslinking agent. Among these carriers, uncharged polysaccharide carriers such as agarose, cellulose, dextran, and pullulan, and crosslinked polysaccharide carriers obtained by crosslinking these with a crosslinking agent, as well as hydrophilic synthetic polymer carriers such as poly(meth)acrylate and polyurethane, and crosslinked hydrophilic synthetic polymer carriers obtained by crosslinking these with a crosslinking agent are preferred because they have hydroxyl groups and can be easily modified with hydrophilic polymers described below.

[0050] Furthermore, in order to suppress nonspecific adsorption of cells, fucose-containing sugar chains, and / or glycoconjugates, the surface of the water-insoluble carrier used in the adsorbent is preferably modified with a hydrophilic polymer, and more preferably the hydrophilic polymer is covalently immobilized on the water-insoluble carrier. Examples of hydrophilic polymers that modify the surface of the water-insoluble carrier include neutral polysaccharides such as agarose, cellulose, dextran, pullulan, and starch, and synthetic polymers containing hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan, and starch are preferred, with dextran and pullulan being more preferred, due to their high hydrophilicity and ease of immobilization on the insoluble carrier surface via covalent bonds. While there are no particular limitations on the molecular weight of dextran and pullulan, those with a number-average molecular weight of 10,000 to 1,000,000 are preferred, as they allow sufficient hydrophilic modification of the insoluble carrier surface. There are no particular restrictions on the shape of the water-insoluble carrier used in the adsorbent, and it may be any of particulate, sponge-like, flat membrane-like, plate-like, hollow, and fibrous. However, particulate carriers are preferred in that they allow efficient cell adsorption to the adsorbent, and spherical particulate carriers are more preferred.

[0051] The average particle size (median diameter) of the water-insoluble carrier used as the adsorbent in the separation and purification method of the present invention, when swollen in water, is preferably 100 μm to 1000 μm, more preferably 100 μm to 500 μm, and even more preferably 150 μm to 300 μm, so that when the adsorbent produced from the carrier is packed into a column, the cells to be separated can be in sufficient contact with the adsorbent surface and cells that do not bind to the adsorbent can pass through the gaps between the adsorbents without stagnation. If the particle size is less than 100 μm, cells that do not bind to the adsorbent will have difficulty passing through the gaps between the adsorbents, resulting in a decrease in cell recovery rate. On the other hand, if the particle size is greater than 1000 μm, contact between the adsorbent-bound cells and the adsorbent surface will be insufficient, resulting in a decrease in the separation efficiency between the adsorbent-bound and non-adsorbent cells. The particle size of the insoluble carrier can be measured, for example, using a precision particle size distribution analyzer (product name "Multisizer 3") manufactured by Beckman Coulter, Inc. Alternatively, the particle size can be determined by taking an image of a graduated glass slide using an optical microscope, then taking images of multiple particles to be measured at the same magnification, measuring the particle sizes of the multiple carriers photographed using a ruler, and calculating the average value. There is no particular restriction on the presence or absence of pores in the water-insoluble carrier used in the adsorbent, and it may be either porous or non-porous.

[0052] Furthermore, the water-insoluble carrier used in the adsorbent of the present invention is preferably a particulate carrier having hydroxyl groups, since this facilitates the introduction of active functional groups for immobilizing the fucose-binding protein used in the adsorbent of the present invention onto the carrier. Furthermore, the water-insoluble carrier used in the adsorbent of the present invention may be a commercially available product, such as Toyopearl (manufactured by Tosoh Corporation), which is made from poly(meth)acrylate, Sepharose (manufactured by GE Healthcare), which is made from agarose, or Cellupher (manufactured by Asahi Kasei Corporation), which is made from cellulose.

[0053] The adsorbent in the separation and purification method of the present invention can be produced by a process including two steps: a step of producing a reactive water-insoluble carrier from a water-insoluble carrier (hereinafter referred to as step X), and a step of immobilizing the fucose-binding protein of the present invention on the reactive water-insoluble carrier by allowing it to react with the carrier (hereinafter referred to as step Y). Steps X and Y are described in detail below.

[0054] Step X is a step of producing a reactive water-insoluble carrier by introducing a reactive functional group for immobilizing the fucose-binding protein of the present invention onto a water-insoluble carrier. The reactive functional group for immobilizing the fucose-binding protein of the present invention onto a water-insoluble carrier is not particularly limited as long as it is a general functional group for protein immobilization, and examples thereof include an epoxy group, a formyl group, a carboxyl group, an active ester group, an amino group, a maleimide group, and a haloacetyl group.

[0055] The method for introducing the functional group into the water-insoluble carrier is not particularly limited as long as it is a common method for introducing a functional group. Examples of the method for introducing an epoxy group include reacting the hydroxyl groups of the water-insoluble carrier with halohydrins such as epichlorohydrin and epibromohydrin, diglycidyl ethers such as ethylene glycol diglycidyl ether, glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, diethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, and resorcinol diglycidyl ether, triglycidyl ethers such as glycerol triglycidyl ether, erythritol triglycidyl ether, and diglycerol triglycidyl ether, and tetraglycidyl ethers such as erythritol tetraglycidyl ether and pentaerythritol tetraglycidyl ether. In addition, when reacting the hydroxyl groups of the water-insoluble carrier with an epoxy group-containing compound, it is preferable to carry out the reaction under basic conditions in order to increase the reaction efficiency.

[0056] Examples of methods for introducing formyl groups into a water-insoluble support include reacting the hydroxyl groups of the water-insoluble support with bifunctional aldehydes such as glutaraldehyde, or reacting the support with an oxidizing agent such as sodium periodate. Another example is a method in which a water-insoluble support into which epoxy groups have been introduced by the above-mentioned method is reacted with a compound such as D-glucamine, N-methyl-D-glucamine, or α-thioglycerol to introduce adjacent hydroxyl groups, and then the resulting water-insoluble support is reacted with an oxidizing agent such as sodium periodate. Examples of methods for introducing carboxyl groups into a water-insoluble support include reacting the hydroxyl groups of the water-insoluble support with a haloacetic acid such as monochloroacetic acid or monobromoacetic acid under basic conditions, as well as reacting the water-insoluble support into which epoxy groups have been introduced by the above-mentioned method with amino acids such as glycine, alanine, aspartic acid, and glutamic acid, amino group-containing carboxylic acids such as β-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid, and sulfur-containing carboxylic acids such as thioglycolic acid and thiomalic acid under basic conditions.

[0057] Another example is a method in which a carboxyl group introduced into a water-insoluble carrier is reacted with N-hydroxysuccinimide in the presence of a condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDC) to convert it into an active ester group, N-hydroxysuccinimide ester. An example of a method for introducing amino groups into a water-insoluble carrier is to react a water-insoluble carrier into which epoxy groups have been introduced by the above-mentioned method with a compound having at least two amino groups, such as ethylenediamine, diethylenetriamine, or tris(2-aminoethyl)amine. An example of a method for introducing maleimide groups into a water-insoluble carrier is to react a water-insoluble carrier having hydroxyl and / or amino groups with a carboxylic acid having a maleimide group, such as 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, or 4-(N-maleimidomethyl)cyclohexanecarboxylic acid, in the presence of a condensing agent such as EDC. Further, a method of reacting an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester of the above-mentioned maleimide group-containing carboxylic acids can be exemplified. Examples of methods for introducing haloacetyl groups into a water-insoluble support include reacting a water-insoluble support having a hydroxyl group or a water-insoluble support into which an amino group has been introduced by the above-mentioned method with an acid halide such as chloroacetic acid chloride, bromoacetic acid chloride, or bromoacetic acid bromide, or reacting a halogenated acetic acid such as chloroacetic acid, bromoacetic acid, or iodoacetic acid in the presence of a condensing agent such as EDC. Further, a method of reacting an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester of the above-mentioned halogenated acetic acid can be exemplified.

[0058] Step Y is a step of immobilizing the fucose-binding protein of the present invention on the reactive water-insoluble carrier produced in step X. The method for immobilizing the fucose-binding protein on the reactive water-insoluble carrier obtained in step X is not particularly limited as long as it is a common protein immobilization method, and examples include a method of immobilizing the protein on a water-insoluble carrier without forming a covalent bond using coordinate bonding or affinity bonding, a method of introducing an active functional group for immobilization into the protein and then reacting the active functional group for immobilization with the carrier to immobilize it on the water-insoluble carrier, and a method of reacting the active functional group for immobilization introduced into the water-insoluble carrier with the protein to form a covalent bond to immobilize it on the water-insoluble carrier. An example of a method for immobilizing a protein on a water-insoluble carrier without forming a covalent bond is a method of using avidin-biotin affinity bonding to immobilize a biotin-introduced protein on a water-insoluble carrier to which avidin has been immobilized, such as Streptavidin Sepharose High Performance (manufactured by GE Healthcare). Examples of methods for introducing biotin into a protein include reacting an amino group of a protein with a biotinylation reagent having an active ester group, such as 9-(biotinamido)-4,7-dioxanonanoic acid-N-succinimidyl, or reacting a mercapto group of a protein with a biotinylation reagent having a maleimide group, such as N-biotinyl-N'-[2-(N-maleimido)ethyl]piperazine hydrochloride. Furthermore, an example of a method for immobilizing a protein by reacting an active functional group for immobilization introduced into the protein with a water-insoluble support to form a covalent bond includes a method in which an amino group of the protein is reacted with an active ester group of a compound having both a maleimide group and an active ester group, such as 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt, to introduce a maleimide group into the protein, and then the resulting mixture is reacted with a water-insoluble support into which a mercapto group has been introduced. Furthermore, examples of methods for immobilizing a protein on a water-insoluble carrier by reacting the protein with an active functional group for immobilization introduced into the water-insoluble carrier include a method of reacting an epoxy group, formyl group, carboxyl group, or active ester group such as N-hydroxysuccinimide ester introduced into the water-insoluble carrier with an amino group on the protein; a method of reacting an amino group introduced into the water-insoluble carrier with a carboxyl group on the protein; and a method of reacting an epoxy group, maleimide group, haloacetyl group, or haloalkyl group introduced into the water-insoluble carrier with a mercapto group on the protein.

[0059] Among these immobilization methods, the method of reacting a formyl group or active ester group introduced into a water-insoluble carrier with an amino group of the protein, and the method of reacting a maleimide group or haloacetyl group introduced into a water-insoluble carrier with a mercapto group of the protein are preferred, as they allow protein immobilization to a water-insoluble carrier in a short time with high yield. The method of reacting a maleimide group or haloacetyl group introduced into a water-insoluble carrier with a mercapto group of the protein is more preferred, as the immobilization reaction can be carried out at a pH close to neutral and protein denaturation can be suppressed. The method of reacting a maleimide group introduced into a water-insoluble carrier with a mercapto group of the protein is even more preferred, as the functional group has high stability.

[0060] The adsorbent of the present invention can be produced by reacting the water-insoluble carrier having the immobilization functional group introduced therein with a fucose-binding protein dissolved in a buffer solution. The buffer solution for dissolving the fucose-binding protein is not particularly limited, and examples include commercially available buffer solutions such as acetate buffer, phosphate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and D-PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries). To improve the efficiency of the immobilization reaction, inorganic salts such as sodium chloride or surfactants such as polyoxyethylenesorbitan monolaurate (Tween 20) may be added to the buffer solution. The reaction temperature and pH when immobilizing a fucose-binding protein on a water-insoluble carrier may be appropriately set within the ranges of 0°C or higher and 50°C or lower, and pH or higher and 10 or lower, taking into consideration the reactivity of the active functional group and the stability of the fucose-binding protein of the present invention. From the viewpoint of preventing inactivation of the fucose-binding protein, the reaction temperature is preferably set within the ranges of 15°C or higher and 40°C or lower, and pH is preferably set within the ranges of pH 5 or higher and 9 or lower.

[0061] The amount of fucose-binding protein immobilized on a water-insoluble carrier can be appropriately determined taking into consideration the binding affinity between the cells to be separated in the separation and purification method of the present invention, and is preferably 0.01 mg to 50 mg, and more preferably 0.05 mg to 30 mg, per mL of water-insoluble carrier. The amount of fucose-binding protein immobilized on a water-insoluble carrier can be adjusted by adjusting the amount of protein used in the immobilization reaction or the amount of active functional groups introduced into the water-insoluble carrier. The amount of fucose-binding protein immobilized on a water-insoluble carrier can be calculated by recovering the immobilization reaction solution and the washing solution after the reaction, determining the amount of unreacted fucose-binding protein, and then subtracting the amount of unreacted fucose-binding protein of the present invention from the amount of fucose-binding protein used in the immobilization reaction.

[0062] Furthermore, as described above, the water-insoluble carrier used in the adsorbent of the present invention preferably has a hydrophilic polymer immobilized thereon by a covalent bond in order to suppress nonspecific adsorption of cells, and therefore, when producing the adsorbent, the hydrophilic polymer can also be immobilized by a covalent bond to the water-insoluble carrier before introducing functional groups for immobilizing the fucose-binding protein of the present invention in step X. The method for immobilizing the hydrophilic polymer by a covalent bond to the water-insoluble carrier is not particularly limited as long as it is a general covalent bond-forming reaction, and an example of such a method is to react hydroxyl groups on the surface of the water-insoluble carrier with an epoxy group-containing compound such as epichlorohydrin, ethylene glycol diglycidyl ether, or 1,4-butanediol diglycidyl ether under basic conditions to introduce epoxy groups into the water-insoluble carrier, and then react the epoxy groups with the hydroxyl groups of the hydrophilic polymer under basic conditions. [Effects of the Invention]

[0063] The present invention can provide a cell separation method that utilizes a cell separation solvent and a cell adsorbent with an animal-free composition.

[0064] Specifically, the present invention provides a method for separating and purifying cells having fucose-containing glycans, such as glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, from a mixture of cells using an animal-free cell separation solvent that does not contain animal-derived components, i.e., an aqueous solution containing a salt of an organic acid and a chelating agent.

[0065] Furthermore, the cell separation method using the cell separation solvent of the present invention can be used for the mass purification of high-quality and high-purity iPS cells for clinical use, and is therefore useful in the medical field, particularly in the field of regenerative medicine. [Brief explanation of the drawings]

[0066] [Figure 1] 1 is a graph showing the cell outflow rate and cell detachment rate in Example 1 and Comparative Example 1. [Figure 2]1 is a graph showing the ratio of the number of detached cells to the number of effluxed cells in Example 1 and Comparative Example 1. [Figure 3] 1 is a graph showing the cell efflux rates in Example 2 and Comparative Example 2. [Example]

[0067] The present invention will be explained in more detail below by giving Preparation Examples, Examples and Comparative Examples, but the present invention is not limited to these.

[0068] Preparation Example 1: Preparation of adsorbent 127Q39L / C72G According to the method described in Examples 12 and 34 of JP 2020-025535 A (Patent Document 4), an adsorbent 127Q39L / C72G was prepared by immobilizing the fucose-binding protein 127Q39L / C72G (amino acid sequence shown in SEQ ID NO: 9) on an insoluble carrier.

[0069] Example 1 Separation of 2102Ep cells using an aqueous solution of organic acids (1) Preparation of a column packed with adsorbent A column was prepared by attaching a 40 μM polyester mesh filter (BioLab) between a 2.5 mL syringe (Terumo) and a 22G needle (Terumo). Next, the adsorbent 127Q39L / C72G prepared in Preparation Example 1 was replaced with MACS buffer, and a 50% suspension of the adsorbent was prepared so that the sedimentation volume of the adsorbent after standing for 12 hours or more was 50%. 1.0 mL of this suspension was added to the prepared column, filling the column with each adsorbent (adsorbent volume: 500 μL). Four columns, columns No. 1 to No. 4, were prepared. Next, column No. 1 was thoroughly filled with an aqueous solution of 0.35 M potassium gluconate with 2 mM EDTA, column No. 2 with an aqueous solution of 0.35 M sodium gluconate with 2 mM EDTA, column No. 3 with an aqueous solution of 0.35 M sodium acetate with 2 mM EDTA, and column No. 4 with an aqueous solution of 0.35 M trisodium citrate with 2 mM EDTA, thereby replacing the adsorbent suspension with each solvent. (2) Cultivation of 2102Ep cells and preparation of cell suspension 2102Ep cells (Embryonal Carcinoma Cells Cl.4 / D3 cells), which are human embryonal carcinoma cells with glycans containing the structure "Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc", were obtained from Cosmobio.

[0070] 2102Ep cells were seeded onto 6 cm diameter adherent culture dishes (Corning) or 10 cm diameter adherent culture dishes (Corning) in D-MEM medium (High Glucose, Fujifilm Wako Pure Chemical Industries) supplemented with 10% FBS (Biological Industries) and antibiotic solution (penicillin-streptomycin solution, Fujifilm Wako Pure Chemical Industries), and cultured at 37°C in a 5% CO2 atmosphere. Next, fluorescent staining of 2102Ep cells using Cell Tracker Orange was performed as follows. After discarding the medium in the Petri dish in which 2102Ep cells were cultured, serum-free RPMI 1640 medium was added and the cells were washed. The serum-free RPMI 1640 medium was then discarded. Next, Cell Tracker Orange dissolved in serum-free RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 μM was added and the cells were cultured at 37°C under a 5% CO2 atmosphere for 1 hour. After discarding the fluorescent reagent solution, D-MEM medium supplemented with 10% FBS and antibiotic solution was added and the cells were cultured at 37°C under a 5% CO2 atmosphere for 1 hour. Next, the D-MEM medium supplemented with 10% FBS and antibiotic solution was discarded, and new D-MEM medium supplemented with 10% FBS and antibiotic solution was added and the cells were cultured overnight at 37°C under a 5% CO2 atmosphere.

[0071] Next, cells were collected and a cell suspension was prepared as follows. The culture medium in the cell culture dish was discarded and D-PBS(-) was added. The cells were then washed and the D-PBS(-) solution was discarded. Next, an appropriate amount of Accutase (Innovative Cell Technology) was added and left for several minutes to detach the 2102Ep cells, which were then collected into four 50 mL tubes (hereafter, the cells dispensed into the tubes will be referred to as Cell No. 1, Cell No. 2, Cell No. 3, and Cell No. 4, respectively). Next, cell No. 1 was suspended in an aqueous solution of 0.35 M potassium gluconate and 2 mM EDTA, cell No. 2 in an aqueous solution of 0.35 M sodium gluconate and 2 mM EDTA, cell No. 3 in an aqueous solution of 0.35 M sodium acetate and 2 mM EDTA, and cell No. 4 in an aqueous solution of 0.35 M trisodium citrate and 2 mM EDTA. The cells were then centrifuged to precipitate and the supernatant was discarded. The cells were then resuspended in the same manner, centrifuged, and the supernatant was discarded, washing the cells. After washing the cells twice, the cells were suspended in appropriate amounts of 0.35 M potassium gluconate and 2 mM EDTA (cell No. 1), 0.35 M sodium gluconate and 2 mM EDTA (cell No. 2), 0.35 M sodium acetate and 2 mM EDTA (cell No. 3), and 0.35 M trisodium citrate and 2 mM EDTA (cell No. 4). The suspensions were then filtered through a cell strainer to prepare 2102Ep cell suspensions stained with Cell Tracker Orange. A portion of the resulting 2102Ep cell suspension was diluted 10-fold and the cell density was calculated using a hemocytometer. Based on this cell density, the amount of cells added to the column was calculated by multiplying the cell density by the volume of cell suspension applied to the column.

[0072] (3) Adsorption and desorption of 2102Ep cells using a column packed with adsorbent Columns filled with adsorbent 127Q39L / C72G were placed vertically, and the cell suspension of 2102Ep cells prepared by the above method was added to each column in a cell solution volume of 0.5 mL so that cell No. 1 was added at a loading amount of 2.1 x 10^5 cells / mL of adsorbent to column No. 1, cell No. 2 was added at a loading amount of 3.2 x 10^5 cells / mL of adsorbent to column No. 2, cell No. 3 was added at a loading amount of 4.8 x 10^5 cells / mL of adsorbent to column No. 3, and cell No. 4 was added at a loading amount of 3.6 x 10^4 cells / mL of adsorbent to column No. 4 (hereinafter, these will be referred to as added cell number -1 to added cell number -4). Next, 1.5 mL of an aqueous solution of 0.35 M potassium gluconate and 2 mM EDTA was added to column No. 1 from the top of the column; 1.5 mL of an aqueous solution of 0.35 M sodium gluconate and 2 mM EDTA was added to column No. 2; 1.5 mL of an aqueous solution of 0.35 M sodium acetate and 2 mM EDTA was added to column No. 3; and 1.5 mL of an aqueous solution of 0.35 M trisodium citrate and 2 mM EDTA was added to column No. 4, and a total of 2 mL of cell fluid was recovered (hereinafter these will be referred to as effluent cell fluid-1 to effluent cell fluid-4, respectively).

[0073] Next, 2.0 mL of an aqueous solution containing 0.35 M potassium gluconate, 0.2 M fucose, and 10 mM EDTA was added to the top of the column (column No. 1), 0.35 M sodium gluconate, 0.2 M fucose, and 10 mM EDTA (column No. 2), 0.35 M sodium acetate, 0.2 M fucose, and 10 mM EDTA (column No. 3), and 0.35 M trisodium citrate, 0.2 M fucose, and 10 mM EDTA (column No. 4) were added to the top of the column, and a total of 2 mL of cell solution was recovered (hereinafter, these will be referred to as detached cell solution-1 to detached cell solution-4, respectively).

[0074] (4) Measurement of cell shedding and detachment rates of 2102Ep cells 500 μL of each of the effluent cell fluid-1 to effluent cell fluid-4 and detached cell fluid-1 to detached cell fluid-4 obtained by the above procedure was taken, and 1.5 mL of MACS buffer was added to make 2 mL. The solution was then dispensed into a 5 mL polystyrene round tube with a cell strainer and cap (manufactured by BD Japan). 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) were added as internal standard beads for cell counting, and 50 μL of 7-AAD was added as a cell viability determination reagent. The cell count was then measured using a cell sorter BD FACSAria (manufactured by BD Japan). The number of cells contained in each of the effluent cell fluids-1 to -4 and the detached cell fluids-1 to -4 was calculated by proportional calculation based on the particle count of the internal standard beads obtained from the dot plot (these cell numbers were designated as effluent cell number-1 to -4 and detached cell number-1 to -4, respectively). The cell effluent rate was calculated by dividing effluent cell number-1 to -4 by added cell number-1 to added cell number-4, respectively, as described in (3). Similarly, the cell detachment rate was calculated by dividing detached cell number-1 to -4 by added cell number-1 to -4, respectively, as described in (3).

[0075] The cell efflux rates were 41.2% for column No. 1, 25.4% for column No. 2, 26.5% for column No. 3, and 11.2% for column No. 4, respectively, which were less than 40%. The adsorption of 2102Ep cells was excellent. The cell detachment rates were 35.2% for column No. 1, 17.1% for column No. 2, 28.8% for column No. 3, and 12.4% for column No. 4. The cell efflux and detachment rates are shown in Table 1 and Figure 1. The ratios of detached cells to effluxed cells calculated from these results were 0.9 for column No. 1, 0.7 for column No. 2, 1.1 for column No. 3, and 1.1 for column No. 4. The ratios of detached cells to effluxed cells are shown in Table 1 and Figure 2. These results showed that the ratio of detached cells to eluted cells was generally 0.7 or higher for all columns, and that the adsorption capacity of 2102Ep cells to the adsorbent was highly maintained regardless of whether 0.35 M potassium gluconate, 0.35 M sodium gluconate, 0.35 M sodium acetate, or 0.35 M trisodium citrate was used as the cell separation solvent. This demonstrated that adsorption separation of cells is possible using solutions that do not contain animal-derived components.

[0076] Comparative Example 1: Separation of 2102Ep cells using aqueous solutions or media other than organic acids Comparative Example 1 relates to a cell separation method using the adsorbent prepared in Example 1 to separate undifferentiated cells by cell adsorption and detachment, using a mannitol aqueous solution, a phosphate buffer-based aqueous solution, and DMEM medium.

[0077] (1) Preparation of a column packed with adsorbent Columns (columns No. 5 to No. 7) packed with the separating agent were prepared in the same manner as in Example 1. Column No. 5 was thoroughly passed through with an aqueous solution of 0.35 M mannitol and 2 mM EDTA, column No. 6 was passed through with an aqueous solution of D-PBS(-) and 2 mM EDTA, and column No. 7 was passed through with an aqueous solution of serum-free DMEM medium and 2 mM EDTA, thereby replacing the adsorbent suspension with each solvent.

[0078] (2) Cultivation of 2102Ep cells and preparation of cell suspension The cell washing procedure was carried out in the same manner as in Example 1, except that for cell No. 5, an aqueous solution of 0.35 M mannitol and 2 mM EDTA was used, for cell No. 6, an aqueous solution of D-PBS(-) and 2 mM EDTA was used, and for cell No. 7, an aqueous solution of serum-free DMEM medium and 2 mM EDTA was used.

[0079] (3) Adsorption and detachment of 2102Ep cells using a column packed with adsorbent Columns filled with adsorbent 127Q39L / C72G were placed vertically, and the cell suspension of 2102Ep cells prepared by the above method was added to each column in a cell solution volume of 0.5 mL so that column No. 5 had a loading amount of 2.0x10^5 cells / mL of adsorbent for cell No. 5, column No. 6 had a loading amount of 6.3x10^5 cells / mL of adsorbent for cell No. 6, and column No. 7 had a loading amount of 6.1x10^5 cells / mL of adsorbent for cell No. 7 (hereinafter, these will be referred to as loaded cell number -5 to loaded cell number -7). Next, 1.5 mL of an aqueous solution of 0.35 M mannitol and 2 mM EDTA was added to column No. 5, 1.5 mL of an aqueous solution of D-PBS(-) and 2 mM EDTA was added to column No. 6, and 1.5 mL of an aqueous solution of serum-free DMEM medium and 2 mM EDTA was added to column No. 7 from the top of the column, and a total of 2 mL of cell fluid was recovered (hereinafter these will be referred to as effluent cell fluid-5 to effluent cell fluid-7, respectively).

[0080] Next, 2.0 mL of an aqueous solution containing 0.35 M mannitol, 0.2 M fucose, and 10 mM EDTA was added to column No. 5 from the top of the column; 2.0 mL of an aqueous solution containing D-PBS(-), 0.2 M fucose, and 10 mM EDTA was added to column No. 6; and 2.0 mL of an aqueous solution containing serum-free DMEM medium, 0.2 M fucose, and 10 mM EDTA was added to column No. 7, and a total of 2 mL of cell solution was recovered (hereinafter, these will be referred to as detached cell solution-5 to detached cell solution-7, respectively).

[0081] (4) Measurement of cell shedding and detachment rates of 2102Ep cells Using the effluent cell fluid-5 to effluent cell fluid-7 and detached cell fluid-5 to detached cell fluid-7 obtained by the above procedure, cell counts were measured in the same manner as in Example 1. The cell efflux rate was calculated by dividing the effluent cell numbers-5 to -7 by the added cell numbers-5 to -7 described in (3), respectively. Similarly, the cell detachment rate was calculated by dividing the detached cell numbers-5 to -7 by the added cell numbers-5 to -8 described in (3), respectively.

[0082] The measurement results showed that the cell efflux rates were 50.2% for column No. 5, 52.2% for column No. 6, and 43.9% for column No. 7, which were generally high at over 40%, demonstrating poor adsorption performance for 2102Ep cells. The cell detachment rates were 8.5% for column No. 5, 20.0% for column No. 6, and 13.5% for column No. 7. The cell efflux rates and cell detachment rates are shown in Table 1 and Figure 1. The ratio of detached cells to effluxed cells calculated from these results was 0.2 for column No. 5, 0.4 for column No. 6, and 0.3 for column No. 7. The ratios of detached cells to effluxed cells are shown in Table 1 and Figure 2. These results showed that the ratio of detached cells to efflux cells was generally 0.4 or less for all columns, and that the number of effluxed 2102Ep cells far exceeded the number of detached cells when any of the following solutions was used as the cell separation solvent: 0.35M mannitol with 2mM EDTA, D-PBS(-) with 2mM EDTA, or serum-free DMEM medium with 2mM EDTA, indicating a reduced adsorption capacity of 2102Ep cells to the adsorbent. This indicates that adsorption and separation of cells using these animal-free solutions is difficult.

[0083] [Table 1]

[0084] Example 2 Separation of iPS cells using an aqueous solution of organic acids (1) Preparation of a column packed with adsorbent A column was prepared by attaching a 40 μM polyester mesh filter (BioLab) between a 2.5 mL syringe (Terumo) and a 22G needle (Terumo). Next, the adsorbent 127Q39L / C72G prepared in Example 1 was replaced with MACS buffer, and a 50% suspension of the adsorbent was prepared so that the sedimentation volume of the adsorbent after standing for 12 hours or more was 50%. 1.0 mL of the suspension was added to the prepared column, filling the column with each adsorbent (adsorbent volume: 500 μL). Three columns, column No. 1 to column No. 3, were prepared. Next, column No. 1 was thoroughly filled with an aqueous solution of 0.18 M potassium gluconate with 2 mM EDTA, column No. 2 with an aqueous solution of 0.18 M sodium gluconate with 2 mM EDTA, and column No. 3 with an aqueous solution of 0.35 M sodium acetate with 2 mM EDTA, thereby replacing the adsorbent suspension with each solvent. (2) Cultivation of 201B7 cells and preparation of cell suspension 201B7 cells, a human iPS cell line having glycans containing the structure "Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc," were obtained from CiRA, Kyoto University, after a patent license agreement and MTA agreement were concluded.

[0085] 201B7 cells were cultured in adherent culture dishes (Corning) as follows. A solution of pre-prepared iMatrix-511 (Nippon) diluted to 3 μg / mL in D-PBS was added to the dish and left at 4°C overnight to coat the culture surface of the dish with iMatrix-511. After discarding the iMatrix-511 solution from the coated dish, the dish was washed with StemFit AK02N medium (Ajinomoto), an iPS cell culture medium. 201B7 cells thawed from a cryovial were suspended in the same medium supplemented with 10 μM lock inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) and seeded. After overnight culture, the StemFit AK02N medium containing Y-27632 was discarded and replaced with StemFit AK02N medium without Y-27632. When the appropriate cell density was reached, the cells were collected and passaged.

[0086] Next, fluorescent staining of 201B7 cells using Cell Tracker Orange was performed as follows. First, the medium in the dish was discarded, and serum-free RPMI 1640 medium was added to rinse the cells, after which the medium was aspirated and discarded. Next, a solution of Cell Tracker Orange dissolved in serum-free RPMI 1640 medium at a final concentration of 10 μM was added, and the cells were cultured for 1 hour at 37°C under a 5% CO2 atmosphere. After discarding the fluorescent reagent solution, StemFit AK02N medium was added, and the cells were cultured for 1 hour at 37°C under a 5% CO2 atmosphere. After discarding the medium, StemFit AK02N medium was added, and the cells were cultured overnight at 37°C under a 5% CO2 atmosphere.

[0087] Next, cells were harvested and a cell suspension was prepared as follows. The cells were rinsed with D-PBS(-) and discarded twice. A detachment solution consisting of a 1:1 mixture of CTS TrypLE Select Enzyme (Thermo Fisher Scientific) and Versene Solution (Thermo Fisher Scientific) was added and incubated at 37°C for 10 minutes under a 5% CO2 atmosphere. After confirming that the cells were detaching in a rounded shape, the cells were detached by repeated pipetting in the detachment solution and collected in a 50 mL tube. The harvested cells were centrifuged to settle, then suspended in D-PBS(-), centrifuged again, and the supernatant discarded. After repeating the cell washing procedure twice, the cells were suspended in D-PBS(-) and filtered through a cell strainer to prepare a cell suspension of 201B7 cells stained with Cell Tracker Orange. A portion of the obtained 201B7 cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Based on this cell density, the amount of cells added to the column was calculated from the cell density x the amount of cell solution applied to the column. (3) Adsorption of 201B7 cells using a column packed with adsorbent Columns packed with adsorbent 127Q39L / C72G were placed upright. 0.1 mL of the 201B7 cell suspension prepared as described above was added to columns 1 through 3 at a cell concentration of 2.1 × 10^6 cells / mL of adsorbent (1.05 × 10^6 cells per 0.5 mL of adsorbent). Next, 1.0 mL of a 0.18 M potassium gluconate solution containing 2 mM EDTA was added to column 1; 0.18 M sodium gluconate solution containing 2 mM EDTA was added to column 2; and 0.35 M sodium acetate solution containing 2 mM EDTA was added to column 3. A total of 1.1 mL of cell suspension was collected (hereafter referred to as "Effluent Cell Suspension-1" to "Effluent Cell Suspension-3"). (4) Measurement of cell efflux and cell detachment rates of 201B7 cells MACS buffer was added to the effluent cell solution-1 through effluent cell solution-3 obtained by the above procedure to make up 2 mL, and the solution was then aliquoted into a 5 mL polystyrene round tube (BD Japan) with a cell strainer and cap. 50 μL of CountBright Absolute Counting Beads (Invitrogen) as internal standard beads for cell counting and 50 μL of 7-AAD as a cell viability test reagent were added, and the cells were counted using a BD FACSAria cell sorter (BD Japan). The number of cells contained in effluent cell solution-1 through effluent cell solution-3 was calculated by proportional calculation based on the particle count of the internal standard beads obtained by dot plot (these cell numbers were designated as effluent cell number-1 through effluent cell number-3, respectively). The cell efflux rate was calculated by dividing effluent cell number-1 through effluent cell number-3 by the number of added cells described in (3).

[0088] The results showed that the cell efflux rates were 1.2% for column No. 1, 1.5% for column No. 2, and 1.0% for column No. 3, with good adsorption of 201B7 cells at less than 1.5%. The cell efflux rates are shown in Table 2 and Figure 3. These results demonstrate that the adsorption capacity of 201B7 cells to the adsorbent was high regardless of whether 0.18 M potassium gluconate, 0.18 M sodium gluconate, or 0.35 M sodium acetate was used as the cell separation solvent. Furthermore, the concentration of organic acid salts or sugar-derived acid salts in the aqueous solution can be adjusted appropriately, demonstrating the feasibility of cell adsorption and separation using these animal-free solutions.

[0089] Comparative Example 2: Separation of iPS cells using aqueous mannitol solution or MACS buffer Comparative Example 2 relates to a cell separation method using the adsorbent prepared in Example 1, in which an aqueous mannitol solution or a MACS buffer solution is used to separate undifferentiated cells by cell adsorption and desorption. (1) Preparation of a column packed with adsorbent Columns (columns Nos. 4 to 7) packed with a separating agent were prepared in the same manner as in Example 2. Column No. 4 was thoroughly filled with an aqueous solution of 0.18 M mannitol and 2 mM EDTA, No. 5 with an aqueous solution of 0.35 M mannitol and 2 mM EDTA, No. 6 with an aqueous solution of 0.70 M mannitol and 2 mM EDTA, and No. 7 with MACS buffer, and the adsorbent suspension was replaced with each solvent. (2) Cultivation of 201B7 cells and preparation of cell suspension The same method as in Example 2 was used. (3) Adsorption of 201B7 cells using a column packed with adsorbent 201B7 cells were loaded onto the columns in the same manner as in Example 2. From the top of the columns, column No. 4 was loaded with 1.0 mL of an aqueous solution of 0.18 M mannitol and 2 mM EDTA, column No. 5 with 1.0 mL of an aqueous solution of 0.35 M mannitol and 2 mM EDTA, column No. 6 with 1.0 mL of an aqueous solution of 0.70 M mannitol and 2 mM EDTA, and column No. 7 with 1.0 mL of MACS buffer. Hereinafter, the eluted cell fluids obtained from columns No. 4 to No. 7 will be referred to as eluted cell fluid-4 to eluted cell fluid-7, respectively. (4) Measurement of cell efflux rate of 201B7 cells MACS buffer was added to the effluent cell fluid-4 to effluent cell fluid-7 obtained by the above procedure to make up 2 mL, and the number of cells contained in the effluent cell fluid-4 to effluent cell fluid-7 was measured in the same manner as in Example 2. The cell efflux rate was calculated by dividing the effluent cell number-4 to effluent cell number-7 by the number of added cells described in (3), respectively.

[0090] The measurement results showed that the cell efflux rates were 2.8% for column No. 4, 2.9% for column No. 5, 2.6% for column No. 6, and 1.8% for column No. 7. The cell efflux rates are shown in Table 2 and Figure 3. These results demonstrate that the cell efflux rates were high (2.6% or higher) when 0.18 M mannitol, 0.35 M mannitol, or 0.70 M mannitol was used as the cell separation solvent, demonstrating the low adsorption capacity of 201B7 cells to the adsorbent. Furthermore, when MACS buffer was used, the cell efflux rate was slightly higher than that of Example 2, even though it contained BSA, an animal-derived component.

[0091] Therefore, it was shown that cell adsorption separation using an appropriately adjusted concentration of mannitol aqueous solution or MACS buffer is difficult to perform cell separation under animal-free conditions due to the low cell adsorption performance and the presence of animal-derived components.

[0092] [Table 2]

Claims

1. A solvent composition used to separate and purify cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, by suspending cells having said sugar chain and cells not having said sugar chain, and contacting the suspension with an adsorbent comprising a fucose-binding protein immobilized on an insoluble carrier, The water-soluble solvent contains a salt of an organic acid and a chelating agent, and does not contain any animal-derived components. the organic acid is citric acid or gluconic acid; A solvent composition characterized in that the concentration of the organic acid is 0.1 mol / L or more and 1.0 mol / L or less.

2. 2. The solvent composition according to claim 1, wherein the salt of the organic acid is a sodium salt or a potassium salt.

3. 3. The solvent composition according to claim 1, wherein the chelating agent is either ethylenediaminetetraacetic acid or ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.

4. A method for separating and purifying cells, comprising the steps of: adsorbing cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc to an adsorbent comprising an insoluble carrier having a fucose-binding protein immobilized thereon, and using the solvent composition according to any one of claims 1 to 3; and then desorbing the cells from the adsorbent using the solvent composition according to any one of claims 1 to 3, which contains fucose.

5. The method according to claim 4, wherein the cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc are human iPS cells.

6. 6. A method for separating and purifying cells according to claim 4 or 5, wherein the fucose-binding protein is any one of the following (a) to (d): (a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or greater. (b) A fucose-binding protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and which has binding affinity to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more. (c) A fucose-binding protein comprising an amino acid sequence containing one or more of the amino acid substitutions listed in (1) to (3) below in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more: (1) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (2) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (3) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (d) A fucose-binding protein comprising the amino acid sequence of the fucose-binding protein of (c) above, in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1, and which has binding affinity to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

7. 6. The method for separating and purifying cells according to claim 4 or 5, wherein the fucose-binding protein is any one of the following (e) to (h): (e) A fucose-binding protein consisting of an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is further added at the N-terminus and an oligopeptide containing cysteine ​​is further added at the C-terminus, wherein X is an integer of 120 or greater. (f) A fucose-binding protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, and further comprising a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, wherein the fucose-binding protein has binding affinity to sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, and X is an integer of 120 or more. (g) A fucose-binding protein comprising an amino acid sequence comprising the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, to which an oligopeptide containing a polyhistidine sequence is further added at the N-terminus and an oligopeptide containing cysteine ​​is added at the C-terminus, and which comprises an amino acid sequence containing one or more of the amino acid substitutions listed in (4) to (6) below, wherein X is an integer of 120 or greater: (4) Substitution of the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) Substitution of the cysteine ​​residue at position 72 of the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from a glycine residue and an alanine residue. (6) Substitution of the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) A fucose-binding protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than positions 39, 65 and 72 of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of (g) above, and further comprising an oligopeptide containing a polyhistidine sequence added to the N-terminus and an oligopeptide containing cysteine ​​added to the C-terminus, and having binding affinity for sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

8. 6. The method for separating and purifying cells according to claim 4 or 5, wherein a hydrophilic polymer is immobilized on a water-insoluble carrier by a covalent bond.

9. 6. A method for separating and purifying cells according to claim 4 or 5, characterized in that an adsorbent packed in a column is used.

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