JP2022151224A - Solvent composition for cell separation - Google Patents

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

The present invention relates to a solvent composition used for separating and purifying cells by suspending at least one or more types of cells and bringing them into contact with an adsorbent, and particularly to solvent compositions containing no animal-derived components.

BC2LCN lectin, which is derived from the N-terminal domain of BC2L-C lectin produced by Gram-negative bacteria (Burkholderia cenocepacia), is a protein having binding affinity to sugar chains containing fucose residues. In addition to the H-type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc) and H-type 3 sugar chain (Fucα1-2Galβ1-3GalNAc) known as undifferentiated sugar chain markers described in Patent Documents 1 and 2, Strong binding to multiple types of sugar chains containing fucose residues such as Lewis Y-type sugar chains (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and Lewis X-type sugar chains (Galβ1-4(Fucα1-3)GlcNAc) It is known to have affinity (Non-Patent Document 2).

In addition, BC2LCN lectin binds to undifferentiated human iPS cells and human ES cells in which H type 1 sugar chains and H type 3 sugar chains are highly expressed, but does not bind to human somatic cells. is known (Non-Patent Document 3). Furthermore, since the BC2LCN lectin has a binding property to the undifferentiated glycan marker, it can be used, for example, for the detection of glycoconjugates containing undifferentiated glycan markers and the detection of undifferentiated cells such as human iPS cells and human ES cells. used (Patent Literature 1 and Patent Literature 2). In addition, it is known that the H-type 1 sugar chain is highly expressed in specific cancer cells as SSEA-5 (Non-Patent Document 4).

Although the BC2LCN lectin has the same level of ability to detect undifferentiated stem cells as anti-Nanog antibodies, which are known antibodies for detecting undifferentiated cells (Patent Document 2), the binding of the BC2LCN lectin and the sugar chains of undifferentiated cells is due to electrostatic interaction. The bond strength is affected by external environment such as solvent and salt concentration. Therefore, depending on the experimental conditions, in the detection of the undifferentiated cells and/or the glycoconjugate containing the undifferentiated sugar chain marker, the binding affinity between the sugar chain and the BC2LCN lectin may be low. A BC2LCN lectin with improved binding affinity to the undifferentiated glycan marker is desired.

As a method for improving the function of a protein, a method of introducing amino acid mutations into the protein by protein engineering techniques to improve the desired function is known. For example, a specific amino acid residue is substituted with another amino acid residue. Fc-binding proteins with improved stability against heat, acid and/or alkali are known (Patent Document 3). In addition, Patent Document 4 discloses a BC2LCN lectin with improved heat stability and binding affinity to sugar chains by amino acid substitution at specific positions using a similar technique (Patent Document 4). Furthermore, a BC2LCN lectin is known that has improved productivity when produced using a microorganism such as Escherichia coli (Patent Document 5).

In addition, as a cell separation and purification method using an adsorbent in which the BC2LCN lectin, which has improved functions such as heat stability and binding affinity to sugar chains and productivity by microbial expression, is immobilized on an insoluble carrier, iPS A method for separating and purifying differentiated cells by removing undifferentiated cells such as cells is known (Patent Document 6), and a method for desorbing and recovering undifferentiated cells adsorbed to an adsorbent is also known (Patent Document 6). Patent document 7).

However, in addition to the above methods, existing methods for separating and purifying cells include a method using magnetic beads (Non-Patent Document 5), cell sorting using a cell sorter (Non-Patent Document 5), and a method using a cell rolling column (Non-Patent Document 5). Reference 6) has been known, but it has been extremely difficult to prepare iPS cells with high quality and purity for clinical use by these methods. For example, culture conditions using bovine serum (hereinafter abbreviated as FBS) or mouse-derived feeder cells as a substitute for FBS have been conventionally used in the culture of iPS cells. However, serum and feeder cells contain unknown factors and there are lot differences, and they may contain viruses and unknown pathogens. Therefore, there has been a demand for a medium with a known composition that uses purified components and synthetic components as much as possible.

In addition, as the cell separation solvent used in the cell separation step, for example, MACS buffer (phosphate buffer (D-PBS(-)) and 2 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) to which 0.5% (w/v) bovine serum albumin (hereinafter abbreviated as BSA), which is an animal-derived component, was added), or fetal bovine serum (hereinafter abbreviated as FBS) and human serum albumin (hereinafter abbreviated as HSA) have been commonly used, but similar to the cell culture process, there is a concern that the quality of cells may deteriorate due to the incorporation of animal-derived components into iPS cells. was Therefore, in the preparation of clinically used iPS cells for regenerative medicine, development of media with a composition that does not contain animal-derived components and allows culture under animal-free conditions is currently underway. A method using TrypLE Select Enzyme (recombinant trypsin), which does not contain animal-derived components, as a stripping solvent in the preparation of , and a method using recombinant HSA added as a cell suspension solvent used during cell separation and purification. Although they have been developed, these reagents are all expensive and have the problem that they cannot be used for preparation of large amounts of cells.

However, at present, for the preparation of iPS cells for regenerative medicine applications, solvent compositions for separating and purifying cells that do not contain animal-derived components, such that a large number of cells can be separated and purified at low cost under animal-free conditions. There was a strong demand for development.

WO2013/065302 WO2013/128914 JP 2011-206046 A JP 2020-025535 JP 2020-058343 JP 2018-134073 JP 2019-000063

Tateno, H. et al., Stem Cells Transl Med. 2013, 2(4):265-273. Sulak, O. et al., Structure. 2010, 18(1):59-72. Tateno, H. et al., J Biol Chem. 2011, 286(23): 20345-20353. Tang, C et al., Nat Biotechnol. 2011, 29(9):829-835. Fong CY et al., Stem Cell Rev Rep. 2009, 5(1):72-80. Mahara, A. et al., J Biomater Sci Polym Ed. 2014, 25(14-15): 1590-601.

An object of the present invention is to provide a solvent composition containing no animal-derived components, which is used for separating and purifying a large amount of cells in a short time under animal-free conditions in the preparation of cells for use in regenerative medicine. Specifically, in a method for separating undifferentiated cells comprising a step of contacting a cell mixture with an adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier, and then peeling off the cells adsorbed on the adsorbent, the undifferentiated A solvent that does not contain any animal-derived components for easy and efficient separation of undifferentiated cells that express markers such as sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the cell surface. It is to provide a composition.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a solvent composition for cell separation containing animal-derived components such as BSA and FBS, which has been conventionally used, can be replaced with a salt of an organic acid and a chelating agent. By using a solvent composition containing, it was found that a large amount of cells can be separated and purified in a short time under animal-free conditions, and the present invention was completed.

That is, the present invention includes the inventions described in [1] to [13] below.
[1] A solvent composition used for separating and purifying cells by suspending at least one or more types of cells and bringing them into contact with an adsorbent, wherein a salt of an organic acid and a chelating agent are added to a water-soluble solvent. A solvent composition characterized by containing and not containing an animal-derived component.
[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 sodium salt or potassium salt.
[4] The solvent composition according to any one of [1] to [3], 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 ethyleneglycolbis(2-aminoethylether)tetraacetic acid.
[6] The adsorbent is an adsorbent in which a fucose-binding protein is immobilized on an insoluble carrier, and after the cells are adsorbed to the adsorbent, the cells are separated and purified by desorbing the cells from the adsorbent. The solvent composition according to any one of [1] to [5], which is used for
[7] Adsorbing cells to the adsorbent using an adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier and the solvent composition according to any one of the above [1] to [6], A method for separating and purifying cells by desorbing cells from an adsorbent using the solvent composition according to any one of claims 1 to 6 containing fucose.
[8] Separating and purifying the cell according to [7] above, wherein the cell has a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc. how to.
[9] Separating the cells according to [8] above, wherein the cells having a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GalNAc are human iPS cells. How to purify.

[10] The method for separating and purifying cells according to any one of [7] to [9] above, wherein the fucose-binding protein is any one of (a) to (d) below.
(a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more. sex protein.
(b) 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 in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, Fucose-binding protein.

[11] The method for separating and purifying cells according to any one of [7] to [9] above, wherein the fucose-binding protein is any one of (e) to (h) below.
(e) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 in the amino acid sequence shown by SEQ ID NO: 1 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.
(g) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of

[12] The method for separating and purifying cells according to any one of [7] to [9], wherein a hydrophilic polymer is covalently immobilized on a water-insoluble carrier.
[13] The method for separating and purifying cells according to any one of [7] to [9], which uses an adsorbent packed in a column.

The present invention will be described in detail below.

The solvent composition of the present invention (hereinafter sometimes abbreviated as the composition of the present invention) is used to separate and purify cells by suspending at least one type of cells and bringing them into contact with an adsorbent. It is a solvent composition that does not contain animal-derived components. The composition of the present invention is particularly suitable for separating and purifying cells by adsorbing cells to an adsorbent in which a fucose-binding protein is immobilized on an insoluble carrier, and then desorbing the cells from the adsorbent. be done.

Since the composition of the present invention does not contain animal-derived components, it is possible not only to reduce the risk of contamination by viruses and unknown pathogens, but also to suppress deterioration in quality due to incorporation of animal-derived components into cells. .

The composition of the present invention is characterized by containing an organic acid salt 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. , dicarboxylic acids such as 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 preferable in that they have low cytotoxicity and can separate and purify cells with high efficiency in the method for separating and purifying cells described below.

Moreover, the salt of the organic acid is preferably a sodium salt or a potassium salt because it is highly water-soluble and does not adversely affect cells. The concentration of the organic acid is not particularly limited as long as it does not kill the cells, but if it is 0.1 mol/L or more and 1.0 mol/L or less, damage to cells due to cell contraction or cell hypertrophy can be suppressed. can.

Furthermore, the composition of the present invention may contain, in addition to the salt of the organic acid described above, a compound that has the effect of promoting the desorption of cells from the adsorbent in the method for separating and purifying cells described later. contains a chelating agent, so that the target cells can be desorbed 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. Tetraacetic acid or ethylene glycol bis(2-aminoethyl ether) tetraacetic acid are preferred.

As described above, the composition of the present invention is suitable for separating and purifying cells by adsorbing cells to an adsorbent in which a fucose-binding protein is immobilized on an insoluble carrier, and then desorbing the cells from the adsorbent. Therefore, the composition of the present invention may contain fucose, which is a compound that dissociates the binding between cells and fucose-binding proteins, in addition to the above-described salts of organic acids and chelating agents. In addition, the composition of the present invention may contain a compound that regulates the osmotic pressure of cells, and a specific example thereof is mannitol. Furthermore, in addition to the aforementioned salts of organic acids, chelating agents, compounds that dissociate the binding between cells and fucose-binding proteins, and compounds that regulate the osmotic pressure of cells, water-soluble A compound that is non-cytotoxic and non-animal derived may be included in the compositions of the present invention.

Next, the method for separating and purifying the 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 uses an adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier and the composition of the present invention described above, so that cells are adsorbed to the adsorbent and then removed from the adsorbent. This is a method for separating and purifying cells by detaching the cells.

Specifically, at least one or more types of cells are brought into contact with an adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier, and then cells not adsorbed to the adsorbent, and the present containing fucose. A method of adding the composition of the invention and obtaining cells detached from the adsorbent can be exemplified. Cells with weak binding to fucose-binding proteins can be desorbed from the adsorbent at low concentrations of fucose, while cells with strong binding to fucose-binding proteins can be desorbed from the adsorbent at high concentrations of fucose. By sequentially adding the composition of the present invention containing, cells having different binding strengths to fucose-binding proteins can be obtained by desorbing from the adsorbent. The fucose concentration in the fucose-containing composition of the present invention, which is used for desorbing the cells adsorbed to the adsorbent, is not particularly limited as long as the concentration does not kill the cells, but is 0.001 mol/L or more. 1.0 mol/L or less is preferable, and 0.01 mol/L or more and 0.5 mol/L or less is more preferable.

In the separation and purification method of the present invention, since an adsorbent obtained by immobilizing a fucose-binding protein to an insoluble carrier, which will be described later, is used, fucose-containing sugar chains to which the fucose-binding protein has binding properties, specifically, H-type Type 1 sugar chain (structure consisting of Fucα1-2Galβ1-3GlcNAc), H type 3 sugar chain (structure consisting of Fucα1-2Galβ1-3GalNAc), Lewis Y sugar chain (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc structure), cells having a Lewis b-type sugar chain (structure consisting of Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), and cells not having these sugar chains are separated and purified to obtain each of them. can be done. Specific examples of cells having sugar chains described above include undifferentiated cells and cancer cells. Undifferentiated cells include human iPS cells and human ES cells. Cancer cells include human embryonic cancer 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. cells. Any of these cells may be, for example, a cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc.

Next, the fucose-binding protein in the separation and purification method of the present invention will be explained. The fucose-binding protein in the separation and purification method of the present invention includes 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), Lewis b-type sugar chains (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), and other proteins having binding properties to fucose-containing sugar chains. It is 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): the amino acid sequence of the recombinant BC2LCN lectin shown in SEQ ID NO: 1 (2 of the amino acid sequence registered with GenPept as accession number WP_006490828; matches the amino acid sequence from th to 156th), wherein X is an integer of 120 or more, or (b): Consists 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 to the Xth amino acid in the amino acid sequence shown by SEQ ID NO: 1, and H type 1 A protein having a binding property to type 3 sugar chains and/or H type 3 sugar chains and X being an integer of 120 or more is expressed as a recombinant protein in a transformant of Escherichia coli. As long as the fucose-binding protein in the separation and purification method of the present invention has binding properties to the fucose-containing sugar chain, particularly to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. , in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown by SEQ ID NO: 1, one or more amino acids may be deleted, substituted or added, for example 15 or less, preferably may have 10 or fewer amino acids deleted, substituted or added.

Moreover, X may be 120 or more and 155 or less, and may be 125 or more and 155 or less. As disclosed in JP-A-2020-25535, the fucose-binding protein in the separation and purification method of the present invention has a plurality of amino acid residues on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1 deleted. Thus, productivity (expression level) can be improved as compared to the case where the amino acid residue is not deleted.

In addition, the fucose-binding protein in the separation and purification method of the present invention is characterized by (i) replacement of the 39th glutamine residue in the amino acid sequence shown by SEQ ID NO: 1 with a leucine residue in terms of improving the stability against heat. , (ii) substitution of the 72nd cysteine residue of the amino acid sequence shown in SEQ ID NO: 1 with one type of amino acid residue selected from glycine and / or alanine residues, (iii) shown in SEQ ID NO: 1 Substitution of the 65th glutamine residue in the amino acid sequence described with a leucine residue.

As disclosed in JP-A-2020-25535, the amino acid substitution described in (i) to (iii) above improves the heat stability of the fucose-binding protein in the separation and purification method of the present invention. be able to. The amino acid substitutions described in (i) to (iii) above are effective in improving the stability against heat whether they are used alone or in combination, but they can further improve the stability against heat. , It is more preferable to combine multiple amino acid substitutions described in (i) to (iii) above.

Furthermore, the fucose-binding protein in the method for separation and purification of the present invention has SEQ ID NO. In the amino acid sequence from the 1st proline to the Xth amino acid in the amino acid sequence shown, one or more amino acid residues are deleted in the region other than the positions substituted by the substitutions (1) to (3). Deletions, substitutions or insertions may be made, for example no more than 15, preferably no more than 10 amino acid residues may be deleted, substituted or inserted.

The fucose-binding protein in the cell separation of the present invention, as long as it has a binding property to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, its N-terminal side and/or The C-terminal side may have an additional amino acid sequence useful for detecting fucose-binding proteins.

The additional amino acid sequences include oligopeptides containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose binding protein, cellulose binding domain, myc tag, FLAG tag and the like. Among these additional amino acid sequences, the productivity is high when produced using E. coli, and the fucose-binding protein can be easily detected by using a fluorescently labeled anti-polyhistidine antibody or anti-GST antibody. , preferably an oligopeptide or GST containing a polyhistidine sequence, more preferably an oligopeptide containing a polyhistidine sequence.

The number of histidine repeat sequences in an oligopeptide containing a polyhistidine sequence is not particularly limited. connectivity may be compromised. Therefore, the length of the repeating sequence of histidines in an oligopeptide containing a polyhistidine sequence is preferably a repeating sequence consisting of 5 to 15 histidines, more preferably a repeating sequence consisting of 5 to 10 histidines. preferable. The position at which the oligopeptide containing the polyhistidine sequence is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. However, it is preferable that the oligopeptide containing the polyhistidine sequence is added to the N-terminal side of the fucose-binding protein in terms of efficient detection with an anti-polyhistidine antibody.

Furthermore, the fucose-binding protein in the separation and purification method of the present invention has a cysteine residue or lysine residue on its N-terminal side and/or C-terminal side, which are useful for immobilizing the fucose-binding protein on a water-insoluble carrier. It may have an additional amino acid sequence consisting of an oligopeptide containing residues (hereinafter referred to as carrier immobilization tag). By immobilizing a fucose-binding protein on a carrier, for example, an undifferentiated cell adsorbent for removing undifferentiated cells such as human iPS cells described in Patent Document 4 can be produced.

The length of the carrier-immobilizing tag 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. do not have. As the tag for carrier immobilization, an oligopeptide consisting of 2 to 10 amino acid residues containing one or more cysteine residues is preferable, since immobilization to a water-insoluble carrier can be performed with high selectivity and high efficiency. includes an oligopeptide consisting of 3 amino acid residues of "Gly-Gly-Cys", an oligopeptide consisting of 5 amino acid residues of "Ala-Ser-Gly-Gly-Cys" and an oligopeptide consisting of 5 amino acid residues of "Gly-Gly-Gly-Ser- An oligopeptide consisting of 7 amino acid residues "Gly-Gly-Cys" can be exemplified. The position at which the oligopeptide containing one or more cysteines is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. , the fucose-binding protein can be efficiently immobilized on a carrier, and the binding activity is less likely to be inhibited because the fucose-binding protein is away from the active center of the fucose-binding protein. The peptide is preferably added to the C-terminal side of the fucose-binding protein.

Specific examples of the fucose-binding protein in the separation and purification method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (amino acid sequences from 1st to 127th in the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 ( Amino acid sequence in which the 72nd cysteine residue of SEQ ID NO: 2 is replaced with a glycine residue), SEQ ID NO: 4 (the 39th glutamine residue of SEQ ID NO: 2 is a leucine residue, and the 72nd cysteine residue is a glycine residue ), SEQ ID NO: 5 (the 39th glutamine residue of SEQ ID NO: 2 is a leucine residue, the 65th glutamine residue is a leucine residue, and the 72nd cysteine residue is a glycine residue ), SEQ ID NO: 6 (amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by SEQ ID NO: 1 and an oligopeptide containing a cysteine residue to the C-terminus), sequence No. 7 (amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by SEQ ID NO: 2 and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 8 (shown by SEQ ID NO: 3) an oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 9 (an amino acid sequence with a polyhistidine sequence at the N-terminus and an oligopeptide containing a cysteine residue at the C-terminus), SEQ ID NO: 9 (poly An oligopeptide containing a histidine sequence, an amino acid sequence obtained by adding an oligopeptide containing a cysteine residue to the C-terminus) and SEQ ID NO: 10 (an oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 5, and a fucose-binding protein represented by any of the amino acid sequences obtained by adding an oligopeptide containing a cysteine residue to the C-terminus.

A signal peptide may be added to the N-terminal side of the fucose-binding protein in the separation and purification method of the present invention to promote efficient expression in the host. Examples of the signal peptide when the host is Escherichia coli include signal peptides such as PelB, DsbA, MalE, and TorT that secrete proteins into the periplasm.

A DNA encoding a fucose-binding protein in the separation and purification method of the present invention can be prepared by a known method. As a method for preparing the DNA, a method of converting the amino acid sequence of the fucose-binding protein into a base sequence in the separation and purification method of the present invention and artificially synthesizing DNA containing the base sequence, or a method of artificially synthesizing DNA containing the base sequence A method of directly and artificially preparing a DNA encoding a fucose-binding protein or a method of preparing it from Burkholderia cenocepacia genomic DNA or the like using a DNA amplification method such as PCR can be exemplified.

In the preparation method, when designing the base sequence, it is preferable to consider the frequency of codon usage in E. coli to be transformed. Ile), ATA, leucine (Leu), CTA, glycine (Gly), GGA, and proline (Pro), CCC, are rare codons (rare codons). Selective conversion is preferred. Analysis of codon usage frequency is performed using a public database (for example, Codon Usage Database on the homepage of Kazusa DNA Research Institute, http://www.kazusa.or.jp/codon/, access date: May 7, 2020). It is also possible by using

In order to transform Escherichia coli with the DNA encoding the fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. It is preferable to insert the DNA into an appropriate position in a vector based on a commonly used bacteriophage, cosmid, plasmid, or the like to form an expression vector, and to transform using the expression vector, since stable transformation can be performed. . Here, the appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the region involved in transmissibility. When inserting the DNA into a vector, it is preferable to insert into the vector in a state of being linked to a functional DNA such as a promoter required for expression.

The vector used as the expression vector is not particularly limited as long as it can stably exist and replicate in the host, and pET vector, pUC vector, pTrc vector, pCDF vector, pBBR vector and the like can be exemplified. Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and λ phage λ PL promoter and λ PR promoter. Transformation of the host Escherichia coli with the expression vector can be carried out by a method commonly used by those skilled in the art. When Escherichia coli W3110 strain or the like is selected, methods described in known literature (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

Next, a method for producing a fucose-binding protein in the present invention will be described. The fucose-binding protein in the present invention is produced by culturing the transformant to produce the fucose-binding protein (hereinafter referred to as the first step) and recovering the fucose-binding protein from the resulting culture. It can be manufactured by a process including two processes (hereinafter referred to as a second process). In the present specification, the culture includes the cultured transformant cells themselves and cell secretions, as well as the medium used for culture. In the first step, the transformant may be cultured in a medium suitable for the culture. For example, when Escherichia coli is used as a host, it is preferable to use Terrific Broth (TB) medium, Luria-Bertani (LB) medium, etc. supplemented with necessary nutrients.

When the expression vector contains a drug resistance gene, selective growth of the transformant becomes possible by adding a drug corresponding to the gene to the medium and carrying out the first step. For example, the expression vector contains a kanamycin resistance gene kanamycin is preferably added to the medium. The culture temperature may be any temperature generally known for the host to be used. It may be determined as appropriate while taking into consideration the production amount of. In addition, the pH of the medium may be within the generally known pH range for the host to be used. For example, when the host is Escherichia coli, the pH is in the range of pH 6.8 to pH 7.4, preferably around pH 7.0. , may be appropriately determined in consideration of the production amount of the fucose-binding protein of the present invention. When an inducible promoter is introduced into the expression vector, the expression of the fucose-binding protein of the present invention can be induced by adding an inducer to the medium under conditions that allow the production of the fucose-binding protein.

Preferred inducers include, for example, isopropyl-β-D-thiogalactopyranoside (IPTG) when using the tac promoter or lac promoter, and the concentration to be added is in the range of 0.005 mM to 1.0 mM, preferably It ranges from 0.01 mM to 0.5 mM. Induction of expression by adding IPTG may be performed under conditions generally known for the host to be 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 may be separated by centrifugation, and the fucose-binding protein of the present invention may be recovered from the resulting culture supernatant. When expressed in the periplasm (including the periplasm in prokaryotes), after collecting the cells by centrifugation, the cells are crushed by adding an enzyme treatment agent, surfactant, etc., and the fucose-binding from the cell lysate. Protein should be collected. Moreover, when it is desired to improve the purity of the fucose-binding protein, a method known in the art may be used, and an example thereof is a separation and purification method using liquid chromatography.

As liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like are preferably used, and a combination of these chromatographies is more preferred. In addition, the purity and molecular weight of the fucose-binding protein of the present invention purified by the above chromatography may be examined using methods known in the art. Examples include SDS polyacrylamide gel electrophoresis (SDS-PAGE) method, Gel filtration chromatography methods can be mentioned.

The binding affinity of the fucose-binding protein to sugar chains in the present invention can be evaluated by Enzyme-linked immunosorbent assay method, surface plasmon resonance method, or the like. As an example, the surface plasmon resonance method will be described. Binding affinity evaluation by the surface plasmon resonance method is performed, for example, using a Biacore T200 instrument (manufactured by GE Healthcare), using a fucose-binding protein as an analyte and a sugar chain (Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1 -3GalNAc structure). Sugar chain-immobilized sensor chips are produced by using biotin-labeled sugar chains to prepare streptavidin-coated sensor chips (Sensor Chip SA, manufactured by GE Healthcare) and dextran-coated sensor chips (Sensor Chip CM5). , GE Healthcare) to which streptavidin has been immobilized in advance. In addition, binding affinity evaluation can be performed using a kinetics analysis program attached to the instrument.

Next, the adsorbent used in the separation and purification method of the present invention will be described. There are no particular restrictions on the raw materials for the water-insoluble carrier used as the adsorbent in the separation and purification method of the present invention. Inorganic carriers such as silica gel and glass on which a thin gold film is vapor-deposited, and polysaccharides such as agarose, cellulose, chitin and chitosan. Water-insoluble polysaccharide carriers used as raw materials 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, polystyrene, etc., and crosslinked synthetic polymer carriers obtained by crosslinking these with a crosslinking agent can be exemplified. Among these carriers, uncharged polysaccharide carriers such as agarose, cellulose, dextran, and pullulan, which have hydroxyl groups and can be easily modified with a hydrophilic polymer to be described later, and their cross-linking agents. Crosslinked crosslinked polysaccharide carriers, 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.

In addition, the surface of the water-insoluble carrier used for the adsorbent is modified with a hydrophilic polymer in order to suppress non-specific adsorption of cells, fucose-containing sugar chains and/or glycoconjugates. More preferably, the hydrophilic polymer is fixed to the water-insoluble carrier by covalent bonding. Hydrophilic polymers that modify the surface of water-insoluble carriers include neutral polysaccharides such as agarose, cellulose, dextran, pullulan and starch, and hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. can be exemplified by synthetic polymers having Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan and starch are preferred, and dextran and pullulan are more preferred, because they are highly hydrophilic and can be easily fixed to the surface of an insoluble carrier by covalent bonding. . Although the molecular weights of dextran and pullulan are not particularly limited, those having a number average molecular weight of 10,000 to 1,000,000 are preferred in terms of sufficient hydrophilic modification of the surface of the insoluble carrier. The shape of the water-insoluble carrier used for the adsorbent is not particularly limited, and may be particulate, sponge-like, flat membrane-like, flat plate-like, hollow, or fibrous. A particulate carrier is preferable, and a spherical particulate carrier is more preferable, because it can be carried out efficiently.

The average particle diameter (median diameter) of the water-insoluble carrier used for the adsorbent in the separation and purification method of the present invention in a state of being swollen in water is the separation target when the adsorbent produced from the carrier is packed in a column. is preferably 100 μm or more and 1000 μm or less, more preferably 100 μm or more and 500 μm or less, in that the cells are in sufficient contact with the adsorbent surface and the cells that do not bind to the adsorbent can pass through the gaps between the adsorbents without stagnation. , more preferably 150 μm or more and 300 μm or less. If the particle size is less than 100 μm, it becomes difficult for cells that do not bind to the adsorbent to pass through the gaps between the adsorbents, resulting in a low cell recovery rate. If the particle size exceeds 1000 μm, the contact between the adsorbent-bound cells and the surface of the adsorbent is insufficient, and the separation efficiency between the adsorbent-bound cells and the non-bound cells is reduced. 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., or the like. Alternatively, after photographing an image of a slide glass with a scale using an optical microscope, images of a plurality of particles to be measured are photographed at the same magnification, and the particle size of the photographed plurality of carriers is measured using a ruler. , can be obtained by calculating the average value thereof. There is no particular limitation on the presence or absence of pores in the water-insoluble carrier used for the adsorbent, and it may be either porous or non-porous.

In addition, the water-insoluble carrier used in the adsorbent of the present invention is a particle having a hydroxyl group in that active functional groups can be easily introduced for immobilizing the fucose-binding protein used in the adsorbent of the present invention on the carrier. A shape carrier is preferred. Furthermore, the water-insoluble carrier used in the adsorbent of the present invention may be a commercially available product. (manufactured by Asahi Kasei Corp.), Cellophia (manufactured by Asahi Kasei Corp.) using cellulose as a raw material, and the like can be used.

The adsorbent in the separation and purification method of the present invention comprises a step of producing a reactive water-insoluble carrier from a water-insoluble carrier (hereinafter referred to as step X), and the addition of the fucose-binding protein of the present invention to the reactive water-insoluble carrier. It can be produced by a process including two processes of acting and immobilizing (hereinafter referred to as process Y). The details of process X and process Y are described below.

Step X is a step of introducing a reactive functional group for immobilizing the fucose-binding protein of the present invention into a water-insoluble carrier to produce a reactive water-insoluble carrier. The reactive functional group for immobilizing the fucose-binding protein of the present invention on a water-insoluble carrier is not particularly limited as long as it is a general functional group for protein immobilization, and includes an epoxy group, a formyl group, a carboxyl group, an active Examples include an ester group, an amino group, a maleimide group, a haloacetyl group and the like.

The method for introducing the functional group into the water-insoluble carrier is not particularly limited as long as it is a general method for introducing the functional group. Halohydrins such as bromohydrin, 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 , resorcinol diglycidyl ether, etc.; triglycidyl ethers, such as glycerol triglycidyl ether, erythritol triglycidyl ether, diglycerol triglycidyl ether; A method of reacting an epoxy group-containing compound such as can be exemplified. Moreover, when reacting the hydroxyl group of the water-insoluble carrier with the epoxy group-containing compound, it is preferable to carry out the reaction under basic conditions in order to increase the reaction efficiency.

Examples of methods for introducing formyl groups into the water-insoluble carrier include a method of reacting a hydroxyl group of the water-insoluble carrier with a bifunctional aldehyde such as glutaraldehyde, and a method of reacting the carrier with an oxidizing agent such as sodium periodate. can do. Alternatively, a water-insoluble carrier into which an epoxy group has been introduced by the above method is reacted with a compound such as D-glucamine, N-methyl-D-glucamine, α-thioglycerol, etc. to obtain a water-insoluble carrier into which adjacent hydroxyl groups have been introduced. , a method of reacting with an oxidizing agent such as sodium periodate. As a method for introducing a carboxyl group into a water-insoluble carrier, in addition to a method of reacting a hydroxyl group of the water-insoluble carrier with a haloacetic acid such as monochloroacetic acid or monobromoacetic acid under basic conditions, an epoxy group was introduced by the method described above. A water-insoluble carrier, 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; A method of reacting with carboxylic acids under basic conditions can be exemplified.

Furthermore, the carboxyl group introduced into the 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). can exemplify a method of derivatization to an N-hydroxysuccinimide ester, which is an active ester group. As a method for introducing an amino group into a water-insoluble carrier, the water-insoluble carrier into which an epoxy group has been introduced by the above method has at least two amino groups such as ethylenediamine, diethylenetriamine, and tris(2-aminoethyl)amine. A method of reacting with a compound can be exemplified. A method for introducing a maleimide group into a water-insoluble carrier includes a water-insoluble carrier having a hydroxyl group and/or an amino group, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, 4-(N-maleimidomethyl ) A method of reacting carboxylic acids having a maleimide group such as cyclohexanecarboxylic acid in the presence of a condensing agent such as EDC can be exemplified. Furthermore, a method of reacting an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester of a carboxylic acid having a maleimide group as described above can be exemplified. Methods for introducing a haloacetyl group into a water-insoluble carrier include, for example, a water-insoluble carrier having a hydroxyl group, a water-insoluble carrier into which an amino group has been introduced by the method described above, and chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, and the like. A method of reacting an acid halide and a method of reacting a halogenated acetic acid such as chloroacetic acid, bromoacetic acid and iodoacetic acid in the presence of a condensing agent such as EDC can be mentioned. Furthermore, a method of reacting the aforementioned N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of halogenated acetic acid can be mentioned.

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 general protein immobilization method. A method of immobilizing a protein on a water-insoluble carrier without forming a covalent bond, and a method of introducing an immobilizing active functional group into a protein and then reacting the immobilizing active functional group with the carrier to form a water-insoluble carrier. Examples include a method of immobilization, and a method of immobilizing on a water-insoluble carrier by reacting an active functional group for immobilization introduced into a water-insoluble carrier with a protein to form a covalent bond. As a method for immobilizing a protein on a water-insoluble carrier without forming a covalent bond, an avidin-biotin affinity bond is used, and a biotin-introduced protein is treated with an avidin such as Streptavidin Sepharose High Performance (manufactured by GE Healthcare). A method of immobilization on an immobilized water-insoluble carrier can be exemplified. Methods for introducing biotin into proteins include a method of reacting a biotinylation reagent having an active ester group such as 9-(biotinamide)-4,7-dioxanonanoic acid-N-succinimidyl with an amino group of the protein; A method of reacting a biotinylation reagent having a maleimido group such as biotinyl-N'-[2-(N-maleimido)ethyl]piperazine hydrochloride with a mercapto group of a protein can be exemplified.
In addition, as a method of immobilizing by reacting an active functional group for immobilization introduced into a protein with a water-insoluble carrier to form a covalent bond, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo -The active ester group of a compound having both a maleimide group and an active ester group, such as N-hydroxysuccinimide ester sodium salt, is reacted with the amino group of the protein to introduce the maleimide group into the protein, and then the water into which the mercapto group is introduced. A method of reacting with an insoluble carrier can be exemplified. Furthermore, as a method of reacting an active functional group for immobilization introduced into a water-insoluble carrier with a protein to immobilize it onto a water-insoluble carrier, epoxy groups, formyl groups, carboxyl groups, and N-hydroxysuccinimide introduced into the water-insoluble carrier can be used. A method of reacting an active ester group such as an ester with an amino group of a protein, a method of reacting an amino group introduced into a water-insoluble carrier with a carboxyl group of a protein, an epoxy group, a maleimide group, a haloacetyl group or a haloalkyl group introduced into a water-insoluble carrier. A method of reacting a group with a mercapto group of a protein can be exemplified.

Among these immobilization methods, a method of reacting a formyl group or an active ester group introduced into a water-insoluble carrier with an amino group of a protein is preferred in that protein can be immobilized onto a water-insoluble carrier in a short period of time with high yield. , and a method of reacting a maleimide group or a haloacetyl group introduced into a water-insoluble carrier with a mercapto group of a protein is preferable, and the immobilization reaction can be carried out at around neutral pH, and protein denaturation can be suppressed. Therefore, the method of reacting the maleimide group or haloacetyl group introduced into the water-insoluble carrier with the mercapto group of the protein is more preferable. More preferred is the method of reacting the mercapto groups.

The adsorbent of the present invention can be produced by reacting the water-insoluble carrier into which the functional group for immobilization has been introduced and the fucose-binding protein dissolved in a buffer solution. The buffer for dissolving the fucose-binding protein is not particularly limited, and may be acetate buffer, phosphate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfone. Commercially available buffers such as acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and D-PBS(−) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) can be exemplified. For the purpose of increasing the efficiency of the immobilization reaction, an inorganic salt such as sodium chloride or a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added to the buffer. The reaction temperature and pH at which the fucose-binding protein is immobilized on the water-insoluble carrier should be 0° C. or higher and 50° C. in consideration of the reactivity of the active functional group and the stability of the fucose-binding protein of the present invention. ° C. or lower, and the pH may be appropriately set within the range of pH 4 or higher and pH 10 or lower, and from the viewpoint of suppressing deactivation of the fucose-binding protein, the reaction temperature is 15 ° C. or higher and 40 ° C. or lower, and the pH is pH 5 or higher. A pH range of 9 or less is preferred.

The amount of fucose-binding protein immobilized on the water-insoluble carrier may be appropriately set in consideration of the binding property between the cells to be separated and the fucose-binding protein in the separation and purification method of the present invention. The amount per insoluble carrier is preferably 0.01 mg or more and 50 mg or less, more preferably 0.05 mg or more and 30 mg or less. In addition, the amount of fucose-binding protein immobilized on the water-insoluble carrier can be adjusted by adjusting the amount of the protein used during the immobilization reaction and the amount of active functional group introduced into the water-insoluble carrier. The amount of fucose-binding protein immobilized on the water-insoluble carrier was determined by recovering the immobilization reaction solution and the post-reaction washing solution to determine the amount of unreacted fucose-binding protein. It can be calculated by subtracting the amount of unreacted fucose-binding protein of the present invention from the amount of protein.

In addition, as described above, the water-insoluble carrier used for the adsorbent in the present invention preferably has a hydrophilic polymer fixed by a covalent bond in order to suppress non-specific adsorption of cells. When producing an agent, before introducing the functional group for immobilizing the fucose-binding protein of the present invention in step X, the hydrophilic polymer can be immobilized on the water-insoluble carrier by covalent bonding. . The method for covalently immobilizing a hydrophilic polymer on a water-insoluble carrier is not particularly limited as long as it is a general covalent bond formation reaction. Epoxy groups are introduced into the water-insoluble carrier by reacting epoxy group-containing compounds such as ether and 1,4-butanediol diglycidyl ether under basic conditions. A method of reacting under conditions can be mentioned.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell separation method using a cell separation solvent and a cell adsorbent having animal-free compositions.

Specifically, according to the present invention, in a mixture of cells having a fucose-containing sugar chain such as a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, the cells having the sugar chain can be separated and purified using an animal-free cell separation solvent containing no animal-derived components, that is, an aqueous solution containing a salt of an organic acid and a chelating agent.

In addition, the cell separation method using the cell separation solvent of the present invention can be used for mass purification of iPS cells with high quality and purity for clinical use, so it is useful in the medical field, especially in the regenerative medicine field.

4 is a graph showing cell outflow rate and cell detachment rate in Example 1 and Comparative Example 1. FIG. 4 is a graph showing the ratio of the number of detached cells to the number of outflow cells in Example 1 and Comparative Example 1. FIG. 4 is a graph showing cell outflow rates in Example 2 and Comparative Example 2. FIG.

The present invention will be described in more detail below with reference to Production Examples, Examples and Comparative Examples, but the present invention is not limited to these.

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

Example 1 Separation of 2102Ep cells using an aqueous organic acid solution (1) Preparation of column filled with adsorbent Polyester with an opening of 40 μM between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G) A column equipped with a mesh filter (manufactured by BioLab) was prepared. Next, after substituting the adsorbent 127Q39L/C72G produced in Preparation Example 1 with MACS buffer, the adsorbent was suspended at 50% so that the sedimentation volume of the adsorbent after standing for 12 hours or longer was 50%. A liquid was prepared, 1.0 mL was added to the prepared column, and each adsorbent was packed in the column (adsorbent capacity: 500 μL). Column NO. Four of 1 to 4 were prepared. Next, column no. NO.1 is an aqueous solution of 0.35M potassium gluconate and 2mM EDTA; 2 is an aqueous solution of 0.35 M sodium gluconate and 2 mM EDTA; 3 is an aqueous solution of 0.35 M sodium acetate added with 2 mM EDTA; In 4, an aqueous solution of 0.35 M trisodium citrate to which 2 mM EDTA was added was sufficiently passed to replace the adsorbent suspension with each solvent.
(2) Culture of 2102Ep cells and preparation of cell suspension 2102Ep cells (Embryonal), which are human embryonal cancer cells having a sugar chain containing a structure consisting of "Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc" Carcinoma Cells Cl.4/D3 cells were obtained from Cosmo Bio.

For 2102Ep cells, D-MEM medium (High Glucose, manufactured by Fujifilm Wako Pure Chemical Industries) supplemented with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries) was used. Cells were seeded in a 6 cm diameter petri dish for adherent culture (manufactured by Corning) or a 10 cm diameter petri dish for adherent culture (manufactured by Corning) and cultured at 37° C. in a 5% CO 2 atmosphere.
Next, fluorescent staining of 2102Ep cells using Cell Tracker Orange was performed by the following method. After discarding the medium in the petri dish in which the 2102Ep cells were being cultured, serum-free RPMI 1640 medium was added to wash the cells, and then the serum-free RPMI 1640 medium was discarded. Next, Cell Tracker Orange dissolved in serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO atmosphere. . After discarding the fluorescent reagent solution, D-MEM medium supplemented with 10% FBS and an antibiotic solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the D-MEM medium supplemented with 10% FBS and an antibiotic solution, a new D-MEM medium supplemented with 10% FBS and an antibiotic solution was added and placed at 37° C. in a 5% CO atmosphere. was cultured overnight.

Next, cell collection and cell suspension preparation were performed by the following methods. After the medium in the petri dish during cell culture was discarded and D-PBS(-) was added, the cells were washed and the D-PBS(-) solution was discarded. Next, an appropriate amount of Accutase (manufactured by Innovative Cell Technology) was added and allowed to stand for several minutes to detach the 2102Ep cells and collect them in four 50 mL tubes (hereafter, the cells dispensed into the tubes are referred to as cell NO. .1, cell No. 2, cell No. 3, and cell No. 4). Cell NO. 1 is an aqueous solution of 0.35 M potassium gluconate with 2 mM EDTA; 2 is an aqueous solution of 0.35 M sodium gluconate with 2 mM EDTA; 3 is an aqueous solution of 0.35 M sodium acetate with 2 mM EDTA, cell NO. 4 after suspending each in an aqueous solution of 0.35 M trisodium citrate and 2 mM EDTA, centrifuging and sedimenting the cells, discarding the supernatant, and resuspending the cells in the same manner , the cells were washed by centrifugation and discarding the supernatant. After repeating the cell washing procedure twice, cell No. 1 is an aqueous solution of 0.35 M potassium gluconate with 2 mM EDTA; 2 is an aqueous solution of 0.35 M sodium gluconate with 2 mM EDTA; 3 is an aqueous solution of 0.35 M sodium acetate with 2 mM EDTA, cell NO. For 4, a cell suspension of 2102Ep cells stained with Cell Tracker Orange was prepared by suspending each using an appropriate amount of an aqueous solution of 0.35 M trisodium citrate and 2 mM EDTA and filtering through a cell strainer. . A portion of the obtained 2102Ep cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column.

(3) Adsorption and Desorption of 2102Ep Cells Using a Column Packed with Adsorbent A cell suspension of 2102Ep cells prepared by the above method was added to the column with the column filled with the adsorbent 127Q39L/C72G standing vertically. NO. 1 has cell NO. 1 added in an amount of 2.1×10̂5/mL-adsorbent, column NO. 2 has cell NO. 2 added in an amount of 3.2×10̂5/mL-adsorbent, column NO. 3 cell no. 3 added in an amount of 4.8×10̂5/mL-adsorbent, column NO. 4 contains cell no. 4 was added to the column at a cell liquid volume of 0.5 mL so that the added amount was 3.6 × 10 ^ 4 cells / mL - adsorbent (hereinafter, these are added cell number -1 to added cell number - 4). Next, from the upper part of the column, column No. Column No. 1 was an aqueous solution of 0.35 M potassium gluconate and 2 mM EDTA. 2 is an aqueous solution of 0.35 M sodium gluconate and 2 mM EDTA; Column No. 3 is an aqueous solution of 0.35 M sodium acetate with 2 mM EDTA added. In 4, 1.5 mL of an aqueous solution of 0.35 M trisodium citrate and 2 mM EDTA was added to collect a total of 2 mL of cell sap (hereafter, these are referred to as effluent cell sap-1 to effluent cell sap- 4).

Next, from the upper part of the column, column No. Column No. 1 was an aqueous solution containing 0.35 M potassium gluconate, 0.2 M fucose and 10 mM EDTA. 2 is an aqueous solution containing 0.35 M sodium gluconate, 0.2 M fucose and 10 mM EDTA; 3 is an aqueous solution containing 0.35 M sodium acetate, 0.2 M fucose and 10 mM EDTA; In 4, 2.0 mL each of an aqueous solution containing 0.35 M trisodium citrate, 0.2 M fucose and 10 mM EDTA was added to collect a total of 2 mL of cell sap (hereinafter referred to as desorbed cell sap-1 ~Desorbed cell sap-4).

(4) Measurement of cell efflux rate and cell desorption rate of 2102Ep cells 500 μL each of effluent cell effluent-1 to effluent cell effluent-4 and desorbed cell effluent-1 to desorbed cell effluent-4 obtained by the above operation Take it out, add 1.5 mL of MACS buffer solution to make it 2 mL, then separate it into a 5 mL polystyrene round tube with a cell strainer cap (manufactured by BD Japan) and use CountBright Absolute Counting Beads (in vitro Gen) and 50 μL of 7-AAD as a reagent for judging cell viability were added, and then the number of cells was measured using a cell sorter BD FACSAria (manufactured by BD Japan). The number of cells contained in each of effluent cell effluent-1 to effluent cell effluent-4 and desorbed cell effluent-1 to desorbed cell effluent-4 was calculated by proportional calculation based on the number of particles of internal standard beads obtained by dot plotting. (This number of cells is defined as the number of outflow cells -1 to -4 and the number of detached cells -1 to the number of detached cells -4. The cell outflow rate is calculated by dividing the number of outflow cells -1 to -4 into (3). It was calculated by dividing the described number of added cells −1 to number of added cells −4.Similarly, the cell detachment rate was obtained by dividing the number of desorbed cells −1 to the number of desorbed cells −4 into the number of added cells described in (3). Calculated by dividing by -1 to 4.

As a result of the measurement, the cell efflux rate was NO. 41.2% in column No. 1; 25.4% on column No. 2; 26.5% on column No. 3, NO. 4 column was 11.2%, which is less than 40%, indicating good adsorption of 2102Ep cells. Also, the cell desorption rate is NO. 35.2% in column No. 1; 17.1% on column No. 2, NO. 28.8% on column No. 3, NO. 4 column was 12.4%. Table 1 and FIG. 1 show the cell outflow rate and cell detachment rate. Also, the ratio of the number of detached cells/the number of outflow cells calculated from these results is NO. 0.9 in a column of 1, NO. 0.7 in the column of 2, NO. 1.1 in the column of 3, NO. 1.1 for 4 columns. Table 1 and FIG. 2 show the ratio of the number of detached cells/the number of outflow cells. From this result, the ratio of the number of exfoliated cells/the number of effluent cells is generally 0.7 or more in any column, and It was found that the ability of 2102Ep cells to adsorb to the adsorbent was maintained at a high level when any aqueous solution of trisodium was used as the cell separation solvent. It was shown that separation is possible.

Comparative Example 1 Separation of 2102Ep cells using an aqueous solution or medium other than an organic acid It relates to a cell separation method using a phosphate buffer-based aqueous solution and DMEM medium.

(1) Production of column filled with adsorbent Columns (column Nos. 5 to 7) filled with a separating agent were prepared in the same manner as in Example 1. column no. 5 is an aqueous solution of 2 mM EDTA added to 0.35 M mannitol; 6 is an aqueous solution prepared by adding 2 mM EDTA to D-PBS(-); In 7, an aqueous solution containing 2 mM EDTA was sufficiently passed through a serum-free DMEM medium to replace the adsorbent suspension with each solvent.

(2) Culture of 2102Ep cells and preparation of cell suspension 5 is an aqueous solution of 2 mM EDTA added to 0.35 M mannitol, cell NO. 6 is an aqueous solution prepared by adding 2 mM EDTA to D-PBS(-), cell NO. 7 was carried out in the same manner as in Example 1, except that an aqueous solution obtained by adding 2 mM EDTA to serum-free DMEM medium was used.

(3) Adsorption and Detachment of 2102Ep Cells Using Adsorbent-Filled Column A column filled with adsorbent 127Q39L/C72G was placed vertically, and the cell suspension of 2102Ep cells prepared by the above method was placed on the column. NO. 5 has cell no. 5 added in an amount of 2.0×10̂5/mL-adsorbent, column NO. 6 cell no. 6 added in an amount of 6.3×10̂5/mL-adsorbent, column NO. 7 has cell no. 7 was added to the column at a cell liquid volume of 0.5 mL so that the added amount was 6.1 × 10^5 cells/mL-adsorbent (hereinafter, these are referred to as added cell number -5 to added cell number -7). described.). Next, from the upper part of the column, column No. For 5, an aqueous solution of 0.35M mannitol and 2mM EDTA, column NO. Column No. 6 is an aqueous solution prepared by adding 2 mM EDTA to D-PBS(-). For 7, 1.5 mL of an aqueous solution containing 2 mM EDTA was added to serum-free DMEM medium to collect a total of 2 mL of cell sap (hereinafter referred to as effluent cell sap-5 to effluent cell sap-7). described.).

Next, from the upper part of the column, column No. 5 was an aqueous solution containing 0.35 M mannitol, 0.2 M fucose and 10 mM EDTA; 6 is an aqueous solution containing D-PBS(-), 0.2 M fucose and 10 mM EDTA; In 7, 2.0 mL each of serum-free DMEM medium and an aqueous solution containing 0.2 M fucose and 10 mM EDTA were added to collect a total of 2 mL of cell fluid (hereinafter referred to as desorbed cell fluid-5 to desorbed referred to as Cell Fluid-7).

(4) Measurement of cell efflux rate and cell detachment rate of 2102Ep cells Performed using effluent cell sap-5 to effluent cell sap-7 and desorbed cell sap-5 to desorbed cell sap-7 obtained by the above operations. Cell counts were determined in the same manner as in Example 1. The cell outflow rate was calculated by dividing the outflow cell number -5 to outflow cell number -7 by the added cell number -5 to the added cell number -7 described in (3), respectively. Similarly, the cell detachment rate was calculated by dividing the detached cell number-5 to detached cell number-7 by the added cell number-5 to the added cell number-8 described in (3), respectively.

As a result of the measurement, the cell efflux rate is column No. 50.2% at column no. 52.2% at column no. 7 is 43.9%, which is generally higher than 40%, and it was clarified that the adsorption performance of 2102Ep cells is low. Also, the cell desorption rate is column No. 8.5% at column no. 20.0% at column no. 7 was 13.5%. Table 1 and FIG. 1 show the cell outflow rate and cell detachment rate. The ratio of the number of detached cells to the number of outflow cells calculated from these results is shown in Column No. 0.2 on 5, column no. 0.4 on column no. 7 was 0.3. Table 1 and FIG. 2 show the ratio of the number of detached cells/the number of outflow cells. From this result, the ratio of the number of desorbed cells to the number of effluent cells is generally 0.4 or less in any column, and an aqueous solution of 0.35 M mannitol with 2 mM EDTA and an aqueous solution of D-PBS(-) with 2 mM EDTA added , an aqueous solution prepared by adding 2 mM EDTA to a serum-free DMEM medium, the number of outflowing 2102Ep cells greatly exceeded the number of detached cells, and the ability of 2102Ep cells to adsorb to the adsorbent was reduced. was found to be declining. Therefore, it was shown that it is difficult to adsorb and separate cells using these animal-free compositions.

Example 2 Separation of iPS Cells Using an Organic Acid Aqueous Solution (1) Preparation of Adsorbent-Filled Column A 40 μM polyester mesh with an opening between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G) A column equipped with a mesh filter (manufactured by BioLab) was prepared. Next, after replacing the adsorbent 127Q39L/C72G prepared in Example 1 with the MACS buffer solution, the adsorbent was suspended at 50% so that the sedimentation volume of the adsorbent after standing for 12 hours or longer was 50%. A liquid was prepared, 1.0 mL was added to the prepared column, and each adsorbent was packed in the column (adsorbent capacity: 500 μL). The columns are column no. 1 to column NO. Three of 3 were prepared. Next, column no. NO.1 is an aqueous solution of 0.18M potassium gluconate and 2mM EDTA; 2 is an aqueous solution of 0.18 M sodium gluconate and 2 mM EDTA; In 3, an aqueous solution of 0.35 M sodium acetate added with 2 mM EDTA was sufficiently passed through to replace the adsorbent suspension with each solvent.
(2) Culturing 201B7 cells and preparing cell suspension After concluding the contract and MTA agreement, it was obtained from Kyoto University CiRA.

The culture of 201B7 cells was performed using a petri dish for adhesion culture (manufactured by Corning) by the following method. A solution prepared in advance by diluting iMatrix-511 (manufactured by Nippi) to 3 μg/mL in D-PBS was added to a petri dish and allowed to stand at 4° C. overnight or longer to coat the culture surface of the petri dish with iMatrix-511. gone. After discarding the iMatrix-511 solution in the coated petri dish, StemFit AK02N medium (manufactured by Ajinomoto Co., Ltd.), which is an iPS cell culture medium, was added and washed. 27632: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was suspended in the same medium to which 10 μM was added and seeded. After overnight culture, the StemFit AK02N medium containing Y-27632 was discarded, and the medium was replaced with StemFit AK02N medium without Y-27632. When the cell density reached an appropriate level, the cells were harvested and subcultured. .

Next, fluorescent staining of 201B7 cells using Cell Tracker Orange was performed by the following method. First, after discarding the medium in the petri dish, serum-free RPMI 1640 medium was added to rinse the cells, and then the medium was aspirated and discarded. Next, a solution obtained by dissolving Cell Tracker Orange in serum-free RPMI 1640 medium at a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, StemFit AK02N medium was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the medium, StemFit AK02N medium was added and cultured overnight at 37°C in a 5% CO2 atmosphere.

Next, cell collection and cell suspension preparation were performed by the following methods. After washing the cells by adding D-PBS (-) to the petri dish and rinsing the cells, discarding the D-PBS (-) was repeated twice, and then the cells were washed with CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific). and Versene Solution (manufactured by Thermo Fisher Scientific) were added at a ratio of 1:1 and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere. After confirming that the cells were detaching in a round shape, the cells were detached by repeating pipetting in the detachment solution and collected in a 50 mL tube. After centrifugation of the collected cells and sedimentation, the cells were suspended in D-PBS(-), centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in D-PBS(-) and filtered using 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. Hereinafter, based on this cell density, the amount of cells added to the column was calculated from cell density × amount of cell solution applied to the column (3) Adsorption of 201B7 cells using a column filled with adsorbent Adsorbent 127Q39L The cell suspension of 201B7 cells prepared by the above method was added to the column NO./C72G in a vertical position. 1 to NO. In 3, 0.1 mL of cell solution was added to the column so that the amount added was 2.1 × 10^6 cells/mL-adsorbent (number of added cells: 1.05 × per 0.5 mL adsorbent). 10^6 pieces). Next, from the upper part of the column, column No. Column No. 1 is an aqueous solution of 0.18 M potassium gluconate and 2 mM EDTA. 2 is an aqueous solution of 0.18 M sodium gluconate and 2 mM EDTA; In 3, a total of 1.1 mL of cell fluid was collected by adding 1.0 mL of an aqueous solution of 0.35 M sodium acetate to which 2 mM EDTA was added (hereinafter, these are referred to as effluent cell fluid-1 to effluent cell fluid- 3).
(4) Measurement of cell efflux rate and cell detachment rate of 201B7 cells. Add MACS buffer to effluent cell effluent-1 to effluent cell effluent-3 obtained by the above operation to make 2 mL, and then add a cell strainer cap. Dispense into a 5 mL polystyrene round tube (manufactured by BD Japan), add 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells, and 50 μL of 7-AAD as a cell viability determination reagent, and then perform a cell sorter. The number of cells was measured using BD FACSAria (manufactured by BD Japan). The number of cells contained in effluent cell sap-1 to effluent cell sap-3 was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting (this number of cells was calculated from the number of effluent cells -1 to The number of outflow cells is defined as −3.The cell outflow rate was calculated by dividing the number of outflow cells −1 to −3 by the number of added cells described in (3).

As a result of the measurement, the cell efflux rate is column No. 1.2% at column no. 1.5% at column no. At 3 m, it was 1.0%, and the adsorption of 201B7 cells was good at 1.5% or less. Cell efflux rates are shown in Table 2 and FIG. From these results, even when any of the aqueous solutions of 0.18 M potassium gluconate, 0.18 M sodium gluconate, and 0.35 M sodium acetate was used as the cell separation solvent, the ability of 201B7 cells to adsorb to the adsorbent was maintained at a high level. It became clear that there was In addition, the concentration of the organic acid salt or sugar-derived acid salt in the aqueous solution can be adjusted as appropriate, and it is possible to adsorb and separate cells using these animal-free composition solutions. was done.

Comparative Example 2 Separation of iPS cells using aqueous mannitol solution or MACS buffer The present invention relates to a cell separation method using a buffer.
(1) Production of column packed with adsorbent Columns (column Nos. 4 to 7) packed with a separating agent were prepared in the same manner as in Example 2.
column no. 4 is an aqueous solution of 2 mM EDTA added to 0.18 M mannitol; 5 is an aqueous solution of 2 mM EDTA added to 0.35 M mannitol; 6 is an aqueous solution of 0.70 M mannitol and 2 mM EDTA; 7 replaced the adsorbent suspension with each solvent by sufficiently passing the MACS buffer solution.
(2) Culture of 201B7 Cells and Preparation of Cell Suspension The same method as in Example 2 was used.
(3) Adsorption of 201B7 cells using column filled with adsorbent 201B7 cells were added to the column in the same manner as in Example 2. From the top of the column, column No. 4 is an aqueous solution of 2 mM EDTA added to 0.18 M mannitol; Column No. 5 is an aqueous solution of 0.35 M mannitol and 2 mM EDTA. Column No. 6 is an aqueous solution of 0.70 M mannitol and 2 mM EDTA. 7 was carried out in the same manner as in Example 2, except that 1.0 mL of MACS buffer was added to each. Column No. below. 4 to NO. The effluent cell sap obtained from 7 are described as effluent cell sap-4 to effluent cell sap-7, respectively.
(4) Measurement of cell efflux rate of 201B7 cells, effluent cell effluent-4 to effluent cell effluent-7 obtained by the above operation were adjusted to 2 mL by adding MACS buffer, followed by the same method as in Example 2. The number of cells contained in effluent cell sap-4 to effluent cell sap-7 was measured. The cell efflux rate was calculated by dividing the number of effluent cells -4 to -7 by the number of added cells described in (3).

As a result of the measurement, the cell efflux rate is column No. 2.8% at column no. 2.9% at column no. 2.6% at column no. 7 was 1.8%. Cell efflux rates are shown in Table 2 and FIG. From this result, even when any of the aqueous solutions of 0.18 M mannitol, 0.35 M mannitol, and 0.70 M mannitol was used as the cell separation solvent, the cell outflow rate was as high as 2.6% or more, and the adsorption of 201B7 cells to the adsorbent. It was found that the adsorption capacity was low. Moreover, when the MACS buffer solution was used, the cell outflow rate was slightly higher than in Example 2 while containing BSA, which is an animal-derived component.

Therefore, adsorption separation of cells using a mannitol aqueous solution or MACS buffer with an appropriately adjusted concentration has low cell adsorption performance and contains animal-derived components when performing cell separation under animal-free conditions. proved difficult.

Claims (13)

A solvent composition used for separating and purifying cells by suspending at least one or more types of cells and bringing them into contact with an adsorbent, the composition containing an organic acid salt and a chelating agent in a water-soluble solvent, A solvent composition that does not contain an animal-derived component. 2. The solvent composition according to claim 1, wherein the organic acid is one selected from acetic acid, citric acid and gluconic acid. 3. The solvent composition according to claim 1, wherein the salt of organic acid is sodium salt or potassium salt. 4. The solvent composition according to any one of claims 1 to 3, wherein the organic acid has a concentration of 0.1 mol/L or more and 1.0 mol/L or less. 5. The solvent composition according to any one of claims 1 to 4, wherein the chelating agent is either ethylenediaminetetraacetic acid or ethyleneglycolbis(2-aminoethylether)tetraacetic acid. The adsorbent is an adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier, and is used to separate and purify cells by adsorbing cells to the adsorbent and then desorbing the cells from the adsorbent. 6. The solvent composition according to any one of claims 1 to 5, wherein the solvent composition is An adsorbent obtained by immobilizing a fucose-binding protein on an insoluble carrier, and the solvent composition according to any one of claims 1 to 6 are used to adsorb cells to the adsorbent, and then fucose is contained. A method for separating and purifying cells by desorbing cells from an adsorbent using the solvent composition according to any one of 1 to 6. The method according to claim 7, wherein the cell is a cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc. 9. The method according to claim 8, wherein the cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc is a human iPS cell. 10. The method for separating and purifying cells according to any one of claims 7 to 9, wherein the fucose-binding protein is any one of (a) to (d) below.
(a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more. sex protein.
(b) 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 in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, Fucose-binding protein. 10. The method for separating and purifying cells according to any one of claims 7 to 9, wherein the fucose-binding protein is any one of (e) to (h) below.
(e) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 in the amino acid sequence shown by SEQ ID NO: 1 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.
(g) to the amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of 10. The method for separating and purifying cells according to any one of claims 7 to 9, wherein a hydrophilic polymer is covalently immobilized on a water-insoluble carrier. 10. The method for separating and purifying cells according to any one of claims 7 to 9, wherein an adsorbent packed in a column is used.

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