JP6699085B2 - Method for purifying Fc binding protein - Google Patents

Description

  The present invention relates to methods for purifying Fc binding proteins using cation exchange chromatography. In particular, the present invention relates to a method of purifying human FcγRIIIa-derived Fc binding protein using cation exchange chromatography with high recovery and high purity.

  Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Fc receptors are classified according to the type of immunoglobulin to which they bind, and include Fcγ receptors that bind to the Fc region of IgG, Fcε receptors that bind to the Fc region of IgE, and Fcα receptors that bind to the Fc region of IgA. Patent Document 1). Further, each receptor is further classified according to the difference in its structure, and in the case of Fcγ receptor, the presence of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb has been reported (Non-Patent Document 1).

Among Fcγ receptors, FcγRIIIa is present on the cell surface of natural killer cells (NK cells) and macrophages, and is an important receptor involved in ADCC (antibody-dependent cellular cytotoxicity) activity, which is important in the human immune system. Is. It has been reported that the affinity between FcγRIIIa and human IgG has a binding constant (KA) showing the strength of binding of 10 ⁷ M ⁻¹ or less (Non-patent Document 2). The amino acid sequence of human FcγRIIIa (SEQ ID NO: 1) has been published in public databases such as UniProt (Accession number: P08637). In addition, the functional domain of the structure of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the position of the cell transmembrane region are also published. Figure 1 shows a structural schematic of human FcγRIIIa. The numbers in FIG. 1 indicate amino acid numbers, and the numbers correspond to the amino acid numbers set forth in SEQ ID NO:1. That is, the 1st methionine (Met) to 16th alanine (Ala) in SEQ ID NO: 1 is the signal sequence (S), and the 17th glycine (Gly) to 208th glutamine (Gln) is the extracellular region. (EC), 209th valine (Val) to 229th valine (Val) are transmembrane regions (TM), and 230th lysine (Lys) to 254th lysine (Lys) are intracellular regions (C). ). It is known that FcγRIIIa strongly binds to IgG1 and IgG3 among human IgG subclasses from IgG1 to IgG4, but weakly binds to IgG2 and IgG4.

  Due to the unexpected immunosuppressive biological properties of Fc receptors discovered in recent years, it is attracting attention as a medicine in the fields of autoimmune diseases or syndromes, transplant rejection and malignant lymphoproliferative growth ( Non-Patent Document 3). Further, the ability of the Fc receptor to adsorb an antibody, which is a function of the Fc receptor, can be utilized as a protein having a function of capturing various chromatography carriers for antibody purification. However, in order to utilize the Fc receptor for the above purpose, it is important to purify the Fc receptor with high purity.

  Regarding the purification of the Fc-binding protein (Fc receptor), the purification of the Fc-binding protein using hydrophobic chromatography is being studied, and the Fc-binding protein adsorbed on the carrier for hydrophobic chromatography is 10% (w/w). By eluting with a buffer solution containing glycerol of v), the Fc-binding protein is highly purified and highly efficient (Patent Document 1). Further, in the purification of the Fc-binding protein using cation exchange chromatography, urea is added to a solution containing the Fc-binding protein applied to the chromatography carrier and an eluate of the Fc-binding protein adsorbed on the carrier. By doing so, adsorption of impurities is suppressed, and highly purified and highly efficient purification is realized (Patent Document 2).

JP, 2011-126827, A JP2012-250945A

J. V. Ravetch et al., Annu. Rev. Immol/Lunol. , 9, 457, 1991 J. Galon et al., Eur. J. Immol/Lunol. , 27, 1928-1932, 1997 Toshiyuki Takai, Jpn. J. Clin. Immol/Lunol. , 28, 318, 2005

  As described above, Patent Documents 1 and 2 disclose the purification of Fc-binding proteins. However, further improvement in purification purity and purification efficiency has been demanded. Further, although the Fc-binding protein examined by the methods disclosed in these patent documents is a protein derived from human FcγRI, when it was similarly examined to a protein derived from another Fc-binding protein, human FcγRIIIa, high purity was obtained. Moreover, highly efficient purification could not be performed.

  Therefore, an object of the present invention is to provide a method for purifying an Fc-binding protein (in particular, an Fc-binding protein derived from human FcγRIIIa) with high purity and high efficiency by using cation exchange chromatography.

  As a result of extensive studies to solve the above-mentioned problems, in a washing solution used in a step of washing a cation exchange chromatography carrier having an Fc-binding protein adsorbed thereon, and an eluate used in a step of eluting the Fc-binding protein from the carrier, The optimum salt concentration was found and the present invention was completed.

That is, the first aspect of the present invention is
(1) adding a solution containing an Fc-binding protein to a carrier for cation exchange chromatography to adsorb the Fc-binding protein on the carrier,
(2) washing the carrier having adsorbed Fc-binding protein with a washing solution,
(3) eluting the Fc-binding protein adsorbed on the carrier using an eluent,
A method for purifying an Fc-binding protein comprising:
In the step (2), after washing with a first washing liquid containing less than 150 mmol/L of sodium chloride, a second washing liquid containing less than 150 mmol/L and a higher concentration of sodium chloride than the first washing liquid is used. It is a step of cleaning with a cleaning liquid,
In the purification method, the step (3) is a step of eluting the Fc-binding protein using an eluent containing 250 mmol/L or more of sodium chloride.

A second aspect of the present invention is that the Fc-binding protein is
A protein containing amino acid residues from at least the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 1 or at least the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 1 Up to and including one or more amino acid residues, and one or more of the amino acid residues are substituted, inserted or deleted by other amino acid residues,
It is the purification method according to the first aspect.

  Hereinafter, the present invention will be described in detail.

  In the present invention, as an example of a solution containing an Fc-binding protein added to a carrier for cation exchange chromatography, a culture solution of a host transformed with an expression plasmid containing a polynucleotide encoding the Fc-binding protein (see below). Or a solution obtained by purifying the culture solution (including the crude purified Fc-binding protein solution described later) using chromatography other than cation exchange chromatography. can give.

  In the present invention, a host transformed with an expression plasmid containing a polynucleotide encoding an Fc-binding protein means an animal cell typified by COS cells or CHO cells, a Bacillus genus (such as Brevibacillus spp. or Paenibacillus spp. Bacteria in a broad sense) and bacteria represented by Escherichia coli, yeasts represented by Saccharomyces, Pichia, Schizosaccharomyces, and filamentous fungi represented by Aspergillus can be exemplified, but Escherichia coli is easy to handle. Is preferably used as the host. When the host is Escherichia coli, the protein may be expressed by culturing the transformant by the method disclosed in JP2012-034591A or JP2013-085531A.

  In the present invention, in order to obtain a crude purified Fc-binding protein solution to be added to a carrier for chromatography from the culture medium of the transformant of the host, it may be appropriately selected depending on the form of expression. For example, when the expressed protein is expressed in the periplasm of host cells, the host cells obtained by centrifuging the culture solution are suspended in an appropriate buffer solution, disrupted by cells (physical disruption, disruption by a drug, etc.), and then centrifuged. It is sufficient to obtain a cell-free extract containing the expressed protein by removing the disruption residue by separation.If the expressed protein leaks from the periplasm of the host cell into the culture supernatant, centrifuge the culture solution. The expressed protein may be recovered from the obtained culture supernatant. When the host cells are disrupted with a drug, for example, the method disclosed in JP2013-252099A or a commercially available extraction reagent such as BugBuster Protein extraction kit (manufactured by Takara Bio Inc.) may be used.

  In the present invention, the carrier for cation exchange chromatography used for purification is not particularly limited as long as it has a cation exchange group such as a carboxymethyl group, a sulfopropyl group, or a sulfonic acid group introduced into the carrier. TOYOPEARL CM-650, TOYOPEARL SP-650, TOYOPEARL GigaCap S-650 (above, Tosoh Corporation make), and CM Sepharose Fast Flow (GE Healthcare company make) are mentioned. When carrying out the purification method of the present invention using the carrier for cation exchange chromatography, the amount of the solution containing Fc-binding protein (applying solution) added to the carrier and the protein adsorption of the carrier The carrier may be packed in an appropriate open column or the like with the amount of the carrier determined by the performance and the like. In addition, the cation exchange chromatography carrier may be prepared by adding a suitable salt-containing buffer solution (Tris-HCl buffer solution, glycine-NaOH buffer solution, phosphate buffer solution, etc.) in advance before adding the apply solution. Equilibrate.

  In the purification method of the present invention, a solution containing an Fc-binding protein (applying solution) is added to a carrier for cation exchange chromatography preliminarily equilibrated with the above-mentioned buffer to adsorb the Fc-binding protein on the carrier. After that, a protein not adsorbed on the carrier is removed using a washing solution before the elution step. At that time, after removing the removal operation using a first cleaning liquid containing less than 150 mmol/L of sodium chloride, a second cleaning liquid containing 150 mmol/L or less and a concentration of sodium chloride higher than that of the first cleaning liquid. By removing with. Specifically, a buffer solution containing sodium chloride having the same ionic strength as the salt contained in the equilibrated buffer solution as the first washing solution, the second washing solution is the first washing solution or more and 150 mmol/L or less. Examples of using a buffer solution containing sodium chloride at a concentration of The concentration of sodium chloride contained in the first cleaning liquid used when performing the removal operation is less than 100 mmol/L, the concentration of sodium chloride contained in the second cleaning liquid is 100 mmol/L or less and more than that of the first cleaning liquid. A high concentration is preferable in that the recovery rate and purity of the Fc-binding protein recovered in the subsequent elution step are further improved. As an example of the preferred embodiment, the buffer solution used for equilibrating the carrier for cation exchange chromatography is a buffer solution having an ionic strength corresponding to sodium chloride in the range of 40 mmol/L to 60 mmol/L, and the first washing solution The concentration of sodium chloride contained in is in the range of 40 mmol/L to 60 mmol/L, and the concentration of sodium chloride contained in the second cleaning liquid is in the range of 80 mmol/L to 100 mmol/L.

  On the other hand, as an eluent for eluting the Fc-binding protein adsorbed on the carrier, a buffer solution containing 250 mmol/L or more of sodium chloride may be used. The concentration of sodium chloride contained in the eluate is more preferably in the range of 300 mmol/L to 400 mmol/L.

In the present invention, the Fc-binding protein means an extracellular region of human FcγRI (specifically, in the case of natural human FcγRI, a region from the 16th glutamine to the 292nd histidine in the amino acid sequence of SEQ ID NO: 5). ), or the extracellular region of human FcγRIIIa (specifically, in the case of natural human FcγRIIIa, the region from the 17th glycine to the 208th glutamine in the amino acid sequence of SEQ ID NO: 1) (Fig. The protein that constitutes 1) may be mentioned. It is not always necessary to use the entire human FcγRI extracellular region or human FcγRIIIa extracellular region, and at least an antibody (immunoglobulin) among proteins (polypeptides) constituting the human FcγRI extracellular region or human FcγRIIIa extracellular region It suffices to include the polypeptide of the region capable of expressing the original function of binding to the Fc region of the. As an example of the human Fc binding protein in the present specification,
(I) a protein containing at least the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 5, or
(Ii) Containing at least the 16th amino acid residue from glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 5, and replacing one or more of the amino acid residues with another amino acid residue , Inserted or deleted proteins,
(Iii) a protein containing an amino acid residue from at least the 17th glycine to the 192nd glutamine in the amino acid sequence of SEQ ID NO: 1,
(Iv) Containing at least the 17th amino acid residue from glycine to the 192nd glutamine in the amino acid sequence of SEQ ID NO: 1, and replacing one or more of the amino acid residues with another amino acid residue , Inserted or deleted proteins,
Can be given. Among them, (iii) and (iv), that is, among the proteins constituting the human FcγRIIIa extracellular region, a protein containing at least a polypeptide capable of expressing the original function of binding to the Fc region of an antibody, or the protein It is preferable to apply the present invention to a protein in which one or more of the amino acid residues constituting the are substituted, inserted or deleted with other amino acid residues.

  Specific examples of (ii) include Fc-binding proteins disclosed in JP2011-206046A and JP2014-027916A.

Further, as a specific example of the above (iv), in the amino acid sequence set forth in SEQ ID NO: 1, the 17th to 192nd amino acid residues are included, and in the 17th to 192nd amino acid residues, the following ( An Fc-binding protein (Japanese Patent Application No. 2014-166883) in which at least any one amino acid substitution among 1) to (40) has occurred.
(1) Substitution of methionine at position 18 of SEQ ID NO: 1 with arginine (2) Substitution of valine at position 27 of SEQ ID NO: 1 with glutamic acid (3) Substitution of phenylalanine at position 29 of SEQ ID NO: 1 with leucine or serine (4) Substitution of leucine at position 30 of SEQ ID NO: 1 with glutamine (5) Substitution of tyrosine at position 35 of SEQ ID NO: 1 with any of aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, and histidine (6) Lysine at position 46 of SEQ ID NO: 1 is replaced with isoleucine or threonine (7) Glutamine at position 48 of SEQ ID NO: 1 is replaced with histidine or leucine (8) Alanine at position 50 of SEQ ID NO: 1 is replaced with histidine (9) Sequence Substitution of 51st tyrosine of No. 1 with aspartic acid or histidine (10) Substitution of 54th glutamic acid of SEQ ID NO: 1 with aspartic acid or glycine (11) Substitution of 56th asparagine of SEQ ID NO: 1 with threonine (12) Substitution of glutamine at position 59 of SEQ ID NO: 1 with arginine (13) Substitution of phenylalanine at position 61 of SEQ ID NO: 1 with tyrosine (14) Substitution of glutamic acid at position 64 of SEQ ID NO: 1 with aspartic acid (15) Substitution of serine at position 65 with arginine (16) Substitution of alanine at position 71 of SEQ ID NO: 1 with aspartic acid (17) Substitution of phenylalanine at position 75 of SEQ ID NO: 1 with leucine, serine or tyrosine (18) Sequence Substitution of 77th aspartic acid of No. 1 with asparagine (19) Substitution of 78th alanine of SEQ ID NO: 1 with serine (20) Substitution of 82nd aspartic acid of SEQ ID NO: 1 with glutamic acid or valine (21) SEQ ID NO: Glutamine at position 90 of 1 was replaced with arginine (22) Asparagine at position 92 of SEQ ID NO: 1 was replaced with serine (23) Leucine at position 93 of SEQ ID NO: 1 was replaced with arginine or methionine (24) 95 of SEQ ID NO: 1 Threonine was replaced with alanine or serine (25) Leucine at position 110 of SEQ ID NO: 1 was replaced with glutamine (26) Arginine at position 115 of SEQ ID NO: 1 was replaced with glutamine (27) Tryptophan at position 116 of SEQ ID NO: 1 Is replaced with leucine (28) Phenylalanine at position 118 of SEQ ID NO: 1 is replaced with tyrosine (29) Lysine at position 119 of SEQ ID NO: 1 is replaced with glutamic acid (30) Glutamic acid at position 120 of SEQ ID NO: 1 is replaced with valine ( 31) Array Substitution of glutamic acid at position 121 of number 1 with aspartic acid or glycine (32) Substitution of phenylalanine at position 151 of sequence number 1 with serine or tyrosine (33) Substitution of serine at position 155 of sequence number 1 (34) The 163rd threonine of No. 1 is replaced with serine (35) The 167th serine of SEQ ID NO: 1 is replaced with glycine (36) The 169th serine of SEQ ID NO: 1 is replaced with glycine (37) The 171st position of SEQ ID NO: 1 Of phenylalanine of (38) asparagine at position 180 in SEQ ID NO: 1 is replaced with any of lysine, serine, and isoleucine (39) Threonine at position 185 of SEQ ID NO: 1 is replaced with serine (40) of SEQ ID NO: 1 Substitution of 192nd glutamine with lysine As another specific example of the above (iv), the amino acid sequence shown in SEQ ID NO: 2 contains the 33rd to 208th amino acid residues, and the 17th to 192nd amino acids. An Fc-binding protein in which at least any one of the following (41) to (111) is substituted in the amino acid residues up to the th position is mentioned, and a more specific example thereof is shown in SEQ ID NO: 3. An Fc-binding protein containing amino acid residues 33 to 208 of the amino acid sequence can be mentioned (Japanese Patent Application No. 2015-047462).
(41) The 45th phenylalanine of SEQ ID NO:2 is replaced with isoleucine or leucine (42) The 55th glutamic acid of SEQ ID NO:2 is replaced with glycine (43) The 64th glutamine of SEQ ID NO:2 is replaced with arginine (44) Substitution of 67th tyrosine of SEQ ID NO: 2 with serine (45) Substitution of 77th phenylalanine of SEQ ID NO: 2 with tyrosine (46) Substitution of 93rd aspartic acid of SEQ ID NO: 2 with glycine (47) of SEQ ID NO: 2 98th aspartic acid is replaced with glutamic acid (48) 106th glutamine of SEQ ID NO: 2 is replaced with arginine (49) 128th glutamine of SEQ ID NO: 2 is replaced with leucine (50) 133rd valine of SEQ ID NO: 2 Is replaced with glutamic acid (51) The lysine at position 135 in SEQ ID NO: 2 is replaced with asparagine or glutamic acid (52) The threonine at position 156 in SEQ ID NO: 2 is replaced with isoleucine (53) The leucine at position 158 in SEQ ID NO: 2 is replaced with glutamine Substitution (54) Substitution of phenylalanine at position 187 of SEQ ID NO: 2 with serine (55) Substitution of leucine at position 191 of SEQ ID NO: 2 with arginine (56) Substitution of serine at position 196 of SEQ ID NO: 2 (57) The 204th isoleucine of No. 2 is replaced by valine (58) The 34th methionine of SEQ ID NO: 2 is replaced by isoleucine, lysine, or threonine (59) The 37th glutamic acid of SEQ ID NO: 2 is replaced by glycine or lysine (60) The 39th leucine of SEQ ID NO:2 is replaced with methionine or arginine (61) The 49th glutamine of SEQ ID NO:2 is replaced with proline (62) The 62nd lysine of SEQ ID NO:2 is replaced with isoleucine or glutamic acid ( 63) Glutamine at the 64th position in SEQ ID NO: 2 was replaced by tryptophan (64) Tyrosine at 67th position in SEQ ID NO: 2 was replaced by histidine or asparagine (65) Glutamic acid at 70th position in SEQ ID NO: 2 was replaced by glycine or aspartic acid ( 66) The 72nd asparagine of SEQ ID NO: 2 is substituted with serine or isoleucine (67) The 77th phenylalanine of SEQ ID NO: 2 is substituted with leucine (68) The 80th glutamic acid of SEQ ID NO: 2 is substituted with glycine (69) sequence The 81st serine of No. 2 was replaced with arginine (70) The 83rd isoleucine of SEQ ID NO: 2 was replaced with leucine (71) The 84th serine of SEQ ID NO: 2 was replaced with proline (72) 85 of SEQ ID NO: 2 Substituting serine with asparagine (73) Substituting alanine at position 87 in SEQ ID NO: 2 with threonine (74) Substituting tyrosine at position 90 in SEQ ID NO: 2 with phenylalanine (75) Arginine with position 91 phenylalanine in SEQ ID NO: 2 Substitution (76) The 93rd aspartic acid of SEQ ID NO:2 is substituted with valine or glutamic acid (77) The 94th alanine of SEQ ID NO:2 is substituted with glutamic acid (78) The 97th valine of SEQ ID NO:2 is methionine and glutamic acid (79) The 98th aspartic acid of SEQ ID NO: 2 is replaced with alanine (80) The 102nd glutamic acid of SEQ ID NO: 2 is replaced with aspartic acid (81) The 106th glutamine of SEQ ID NO: 2 is replaced with leucine ( 82) The 109th leucine of SEQ ID NO:2 was replaced with glutamine (83) The 117th glutamine of SEQ ID NO:2 was replaced with leucine (84) The 119th glutamic acid of SEQ ID NO:2 was replaced with valine (85) SEQ ID NO:2 At position 121 in arginine is substituted (86) Proline at position 130 in SEQ ID NO: 2 is replaced by leucine (87) Lysine at position 135 in SEQ ID NO: 2 is replaced by tyrosine (88) Glutamic acid at position 136 in SEQ ID NO: 2 Is substituted with valine (89) Histidine at position 141 of SEQ ID NO: 2 is replaced with glutamine (90) Serine at position 146 of SEQ ID NO: 2 is replaced with threonine (91) Lysine at position 154 of SEQ ID NO: 2 is replaced with arginine ( 92) Glutamine at position 159 in SEQ ID NO: 2 was replaced with histidine (93) Glycine at position 163 in SEQ ID NO: 2 was replaced with valine (94) Lysine at position 165 in SEQ ID NO: 2 was replaced with methionine (95) SEQ ID NO: 2 Phenylalanine at position 167 is replaced with tyrosine (96) Histidine at position 169 of SEQ ID NO: 2 is replaced with tyrosine (97) Tyrosine at position 174 of SEQ ID NO: 2 is replaced with phenylalanine (98) Lysine at position 177 of SEQ ID NO: 2 Is replaced with arginine (99) The 185th serine of SEQ ID NO:2 is replaced with glycine (100) The 194th serine of SEQ ID NO:2 is replaced with arginine (101) The 196th asparagine of SEQ ID NO:2 is replaced with lysine ( 102) Substitution of 201st threonine of SEQ ID NO: 2 with alanine (103) Substitution of 203th asparagine of SEQ ID NO: 2 with isoleucine or lysine (104) Substitution of 207th threonine of SEQ ID NO: 2 with alanine (105) sequence The 94th alanine of number 2 is Substitution with phosphorus (106) Substitution of 98th aspartic acid of SEQ ID NO: 2 with glutamic acid (107) Substitution of 117th glutamine of SEQ ID NO: 2 with arginine (108) Substitution of 156th threonine of SEQ ID NO: 2 with isoleucine ( 109) The 174th tyrosine of SEQ ID NO:2 is replaced with histidine (110) The 181st lysine of SEQ ID NO:2 is replaced with glutamic acid (111) The 203rd asparagine of SEQ ID NO:2 is replaced with aspartic acid or tyrosine. Another specific example of iv) includes the 17th to 192nd amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1, and the 17th to 192nd amino acid residues have the following ( An Fc-binding protein in which at least one amino acid substitution among the naturally occurring amino acid substitutions from (112) to (115) has occurred.
(112) Substitution of leucine at position 66 of SEQ ID NO: 1 with histidine or arginine (113) Substitution of glycine at position 147 of SEQ ID NO: 1 with aspartic acid (114) Substitution of tyrosine at position 158 of SEQ ID NO: 1 with histidine (115) ) Phenylalanine is substituted for the 176th valine of SEQ ID NO: 1 In the purification method of the present invention, in order to quantify the Fc-binding protein contained in the fraction eluted from the column, it is possible to stably and efficiently use a conventionally known method. The method may be appropriately selected from among quantifiable methods, but an analysis method by an ELISA method (enzyme-linked immunosorbent method) is preferable.

  The Fc-binding protein obtained by the purification method of the present invention can be used for various applications such as pharmaceuticals, clinical test agents, biosensors, and affinity ligands (separating agents). The form and purity at the time of use differ depending on the intended use, and the Fc-binding protein obtained by the purification method of the present invention may be used as it is, or may be a highly purified product, or an intermediate thereof. You may use the thing of the purification degree of.

  The present invention uses (1) a step of adsorbing a solution containing an Fc-binding protein to a carrier for cation exchange chromatography to adsorb the Fc-binding protein on the carrier, and (2) a washing solution, A method for purifying an Fc-binding protein, comprising the steps of washing the carrier having adsorbed the Fc-binding protein, and (3) eluting the Fc-binding protein adsorbed to the carrier using an eluent. After the step (2) is washed with a first washing liquid containing less than 150 mmol/L sodium chloride, a second washing liquid containing 150 mmol/L or less and a higher concentration of sodium chloride than the first washing liquid is used. The step (3) is characterized in that the Fc-binding protein is eluted with an eluent containing 250 mmol/L or more of sodium chloride. According to the present invention, Fc-binding protein (particularly Fc-binding protein derived from human FcγRIIIa) can be purified with high recovery and high purity by one-step column chromatography operation.

  In addition, since the purification method of the present invention is a method that can be applied regardless of the amount of the solution containing the Fc-binding protein to be introduced into the carrier (applying amount), it is of course applicable to the purpose of analyzing Fc-binding protein, It can also be applied to one step of industrial Fc binding protein production.

  The Fc-binding protein obtained by the purification method of the present invention can be used as it is or after further purification for various applications such as pharmaceuticals, clinical test agents, biosensors, or affinity ligands (separating agents).

The figure which shows the structure of human FcγRIIIa. The result of confirming the purity of the fraction obtained in the elution step in Example 2 (chromatogram of size exclusion chromatography). The result of confirming the purity of the fraction obtained in the elution step in Comparative Example 1 (chromatogram of size exclusion chromatography).

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

Example 1 Preparation of Fc-binding protein A solution containing Fc-binding protein used in the following examples was prepared by the following method.
(1) An expression vector pTrcFcR9T8-R1 containing a polynucleotide (SEQ ID NO: 4) encoding an Fc-binding protein consisting of the sequence shown in SEQ ID NO: 3 was prepared by the method disclosed in JP-A-2014-223064. Escherichia coli was transformed with the expression vector. The Fc-binding protein consisting of the sequence set forth in SEQ ID NO: 3 contains the amino acid residues 33 to 208 of the amino acid sequence set forth in SEQ ID NO: 2, and the amino acid residues 33 to 208 are the same. It is an Fc-binding protein having the following amino acid substitutions (I) to (IV) in the group (Japanese Patent Application No. 2015-047462).
(I) 45th phenylalanine of SEQ ID NO: 2 is replaced with isoleucine (II) 64th glutamine of SEQ ID NO: 2 is replaced with arginine (III) 133rd valine of SEQ ID NO: 2 is replaced with glutamic acid (IV) SEQ ID NO: The recombinant Escherichia coli obtained by substituting the 187th phenylalanine of 2 with serine (2) was cultured according to the method disclosed in JP2012-034591A and JP2013-085531A, thereby binding Fc. Sex protein was expressed.
(3) After recovering the bacterial cells from the culture solution of recombinant Escherichia coli, the extract (1 mmol/L EDTA, 40 mmol/L sodium chloride, 2 mmol/L magnesium sulfate, 250 Unit/L Benzonase (manufactured by Merck), 20 mmol/mol containing 0.0005 (w/v)% lysozyme, 0.5 (w/v)% Triton X-100 (trade name), and 0.001 (w/v)% sodium deoxycholate. The protein expressed in the cells was extracted with L phosphate buffer (pH 6.0).
(4) The supernatant was recovered from the bacterial cell extract obtained in (3) by centrifugation to obtain an Fc-binding protein extract.
(5) Since the extract obtained in (4) was added to the carrier for cation exchange chromatography, the ionic strength (electrical conductivity) was adjusted to an ionic strength corresponding to a sodium chloride concentration of 50 mmol/L. .

Example 2 Purification of Fc-binding protein (1) The Fc-binding protein extract obtained in (5) of Example 1 was treated with 20 mmol/L phosphate buffer (pH 6) containing 50 mmol/L sodium chloride. .0) was applied in advance to a cation exchange chromatography carrier TOYOPEARL CM-650M (manufactured by Tosoh Corporation) to adsorb the Fc-binding protein to the carrier.
(2) Contaminant impurities were removed by washing the carrier having adsorbed the Fc-binding protein with 20 mmol/L phosphate buffer (pH 6.0) containing 50 mmol/L sodium chloride. Cleaning step).
(3) Contaminant impurities were removed by further washing with 20 mmol/L phosphate buffer (pH 6.0) containing 100 mmol/L sodium chloride added (second washing step).
(4) The Fc-binding protein was eluted by applying 20 mmol/L phosphate buffer (pH 6.0) containing 350 mmol/L sodium chloride to the carrier having the Fc-binding protein adsorbed thereon. (Elution step).
(5) The amount of Fc-binding protein contained in the fraction obtained in the second washing step and the fraction obtained in the elution step was obtained using the ELISA method disclosed in JP-A-2014-223064. Then, the recovery rate of Fc-binding protein was calculated by dividing the amount of Fc-binding protein contained in each fraction by the amount of added Fc-binding protein. The purity of the Fc-binding protein was calculated by size exclusion chromatography (TSKGel G3000SWXL, manufactured by Tosoh Corporation).

Examples 3 to 5 and Comparative Examples 1 to 4
20 mmol/L used in the first washing step described in Example 2(2), the second washing step described in Example 2(3), and the elution step described in Example 2(4). The Fc-binding protein was purified in the same manner as in Example 2 except that the concentrations of sodium chloride contained in the phosphate buffer (pH 6.0) were changed to those shown in Table 1. In Comparative Examples 1 and 2, the second cleaning step is skipped.

実施例２から５ならびに比較例１から４の結果を表２にまとめて示す。第二の洗浄工程で用いる洗浄液に含まれる塩化ナトリウム濃度を１５０ｍｍｏｌ／Ｌ以下かつ第一の洗浄工程で用いる洗浄液に含まれる塩化ナトリウム濃度（５０ｍｍｏｌ／Ｌ）以上とし、溶出工程で用いる洗浄液に含まれる塩化ナトリウム濃度を２５０ｍｍｏｌ／Ｌ以上とすることで、溶出工程で得られる画分におけるＦｃ結合性タンパク質の回収率が６５％以上かつ純度も８５％以上となり、Ｆｃ結合性タンパク質を高回収率かつ高純度に精製できることがわかる（実施例２から５）。なお実施例２において、溶出工程で得られる画分の純度を確認した結果（クロマトグラム）を図２に示す。

The results of Examples 2 to 5 and Comparative Examples 1 to 4 are summarized in Table 2. The concentration of sodium chloride contained in the cleaning solution used in the second cleaning step is 150 mmol/L or less and the concentration of sodium chloride contained in the cleaning solution used in the first cleaning step is 50 mmol/L or more, and contained in the cleaning solution used in the elution step. By setting the sodium chloride concentration to 250 mmol/L or more, the Fc-binding protein recovery rate in the fraction obtained in the elution step is 65% or more and the purity is 85% or more, and the Fc-binding protein recovery rate is high. It can be seen that it can be purified to a high degree of purity (Examples 2 to 5). In addition, in Example 2, the result (chromatogram) of confirming the purity of the fraction obtained in the elution step is shown in FIG.

  On the other hand, when the second washing step is skipped, it can be seen that the purity (Comparative Example 1) or recovery rate (Comparative Example 2) of the Fc-binding protein in the fraction obtained in the elution step decreases. Even if the second washing step is performed, if the sodium chloride concentration in the washing solution used in the second washing step is 200 mmol/L or more, the Fc-binding protein is eluted in the fraction obtained in the second washing step. Therefore, it can be seen that the recovery rate of Fc-binding protein (Comparative Examples 3 and 4) in the fraction obtained in the elution step is significantly reduced. In Comparative Example 1, the result (chromatogram) of confirming the purity of the fraction obtained in the elution step is shown in FIG.

Claims (1)

(1) A step of adsorbing an Fc-binding protein on the carrier by adding a solution containing an Fc-binding protein to the carrier for cation exchange chromatography,
(2) washing the carrier having adsorbed Fc-binding protein with a washing solution,
(3) eluting the Fc-binding protein adsorbed on the carrier using an eluent,
A method for purifying an Fc-binding protein comprising:
Step (2) is washed with a first washing solution containing sodium chloride of 5 0 mmol / L, a step of washing with a second wash solution containing sodium chloride 100 mmol / L,
Wherein (3) the step of, Ri step der eluting the Fc binding protein with an elution solution containing the following sodium chloride 300 mmol / L or more 400 mmol / L,
Fc binding protein
(I) a protein containing an amino acid residue from at least the 33rd glycine to the 208th glutamine in the amino acid sequence of SEQ ID NO: 3 , or
(Ii) Amino acid from at least the 33rd glycine to the 208th glutamine in the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence in which at least one amino acid substitution of the following (a) to (d) occurs: it is including protein residues,
METHODS;
(A) Replacing the leucine corresponding to the 66th position in SEQ ID NO: 1 with histidine or arginine
(B) Glycine corresponding to the 147th position of SEQ ID NO: 1 is replaced with aspartic acid
(C) Tyrosine corresponding to the 158th position of SEQ ID NO: 1 is replaced with histidine
(D) The valine corresponding to the 176th position of SEQ ID NO: 1 was substituted with phenylalanine.

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