JP7676866B2 - Stock solution of human FcγRIIIa - Google Patents

Description

The present invention relates to a storage solution of FcγRIIIa. In particular, the present invention relates to a storage solution of human FcγRIIIa that improves the solubility of human FcγRIIIa and allows storage without precipitation.

Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Each molecule recognizes a single or the same group of immunoglobulin isotypes through a recognition domain on the Fc receptor, which belongs to the immunoglobulin superfamily. This determines which accessory cells are mobilized in the immune response. Fc receptors can be further classified into several subtypes, including Fcγ receptors, which are receptors for IgG (immunoglobulin G), Fcε receptors that bind to the Fc region of IgE, and Fcα receptors that bind to the Fc region of IgA. Each receptor is further classified, and the existence of Fcγ receptors has been reported as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb (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), which is an important activity in the human immune system. It has been reported that the affinity between this FcγRIIIa and human IgG is 10 ⁻⁷ mol/L or less, which indicates the strength of binding (K _D value 2). It is also known that the binding ability between FcγRIIIa and an antibody varies depending on the sugar chain structure of the antibody (Non-Patent Document 3).

The amino acid sequence of native human FcγRIIIa (SEQ ID NO: 1) has been published in public databases such as UniProt (Accession number: P08637). The structural functional domains of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the position of the cell membrane-transducing region have also been published. Figure 1 shows a schematic diagram of the structure of human FcγRIIIa. The numbers in Figure 1 indicate amino acid numbers, and these numbers correspond to the amino acid numbers in SEQ ID NO: 1. That is, in SEQ ID NO: 1, the sequence from the 1st methionine (Met) to the 16th alanine (Ala) is the signal sequence (S), the sequence from the 17th glycine (Gly) to the 208th glutamine (Gln) is the extracellular domain (EC), the sequence from the 209th valine (Val) to the 229th valine (Val) is the transmembrane domain (TM), and the sequence from the 230th lysine (Lys) to the 254th lysine (Lys) is the intracellular domain (C). It is known that FcγRIIIa binds strongly to IgG1 and IgG3, particularly among the human IgG subclasses ranging from IgG1 to IgG4, while its binding to IgG2 and IgG4 is weak.

Because human FcγRIIIa recognizes the glycan structure of antibodies, an adsorbent obtained by immobilizing human FcγRIIIa on an insoluble carrier can separate antibodies based on differences in binding affinity based on their glycan structure (Patent Documents 1 and 2). Because the glycan structure of an antibody is closely related to the efficacy and stability of antibody pharmaceuticals, it is extremely important to control the glycan structure when producing antibody pharmaceuticals. For this reason, the adsorbent is useful for process analysis and separation during the production of antibody pharmaceuticals.

Human FcγRIIIa can be produced with high purity and efficiency by culturing recombinant Escherichia coli into which a gene encoding human FcγRIIIa has been inserted, and subjecting the resulting culture medium to cation exchange chromatography (Patent Document 3). However, there is a problem that human FcγRIIIa contained in the elution fraction of the chromatography precipitates, particularly in human FcγRIIIa that has improved alkaline resistance through amino acid substitution.

JP 2015-086216 A JP 2016-169197 A JP 2016-183113 A

Takai. T. , Jpn. J. Clin. Immunol. , 28, 318-326, 2005 J. Galon et al., Eur. J. Immunol. , 27, 1928-1932, 1997 C. L. Chen et al., ACS Chem. Biol. , 12, 1335-1345, 2017

The objective of the present invention is to provide a storage solution that improves the solubility of human FcγRIIIa (particularly human FcγRIIIa with improved alkaline resistance through amino acid substitution) and allows stable storage without precipitation.

As a result of intensive research to solve the above problems, the inventors discovered that adding an appropriate concentration of arginine and/or its analog to a storage solution containing human FcγRIIIa improves the solubility of human FcγRIIIa in the storage solution and prevents precipitation, which led to the completion of the present invention.

That is, the present invention includes the following aspects [1] to [5].

[1] A storage solution of human FcγRIIIa, the storage solution containing 0.2 mol/L or more and 1 mol/L or less of arginine and/or its analogue.

[2] The preservation solution according to [1], further comprising glycerol at 3 (w/v)% or more and 45 (w/v)% or less.

[3] A method for stabilizing human FcγRIIIa, comprising a step of contacting human FcγRIIIa with the preservation solution described in [1] or [2].

[4] A method for producing human FcγRIIIa, comprising the steps of applying a solution containing human FcγRIIIa to a column packed with a chromatography carrier to adsorb human FcγRIIIa to the carrier, applying an elution buffer to the column to elute human FcγRIIIa adsorbed to the carrier, recovering a fraction containing the eluted human FcγRIIIa, and storing the recovered fraction, wherein the storing step is a step of storing in the storage solution described in [1] or [2].

[5] The preservation solution according to [1] or [2], wherein the human FcγRIIIa is a polypeptide selected from the group consisting of (a) and (c) below:
(a) a polypeptide comprising at least the amino acid residues from the 24th glycine to the 199th glutamine of the amino acid sequence set forth in SEQ ID NO:2;
(b) a polypeptide having an amino acid sequence including at least the amino acid residues from glycine at position 24 to glutamine at position 199 of the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid sequence includes one or more substitutions, deletions, insertions, or additions of amino acid residues at one or more positions within the amino acid residues at positions 24 to 199, and having antibody-binding activity;
(c) A polypeptide comprising at least the amino acid residues from the 24th glycine to the 199th glutamine of the amino acid sequence set forth in SEQ ID NO:2, and having a homology of 80% or more to the amino acid sequence from the 24th to the 199th amino acid, and having antibody binding activity.

The present invention is described in detail below.

The human FcγRIIIa storage solution of the present invention is characterized in that the solution containing human FcγRIIIa contains arginine and/or its analogs at a final concentration of 0.2 mol/L to 1 mol/L, and the solubility of human FcγRIIIa present in the solution is improved, thereby suppressing the occurrence of precipitation. It is preferable that the arginine and/or its analogs contained in the human FcγRIIIa storage solution be at a final concentration of 0.3 mol/L to 0.8 mol/L.

The arginine is preferably L(+)-arginine. Examples of arginine analogues include L(+)-arginine hydrochloride, Nα-carbobenzoxy-L(+)-arginine, Nα-tosyl-L(+)-arginine, Nα-(2,4-dinitrophenyl)-L(+)-arginine, Nα-(tert-butoxycarbonyl)-L(+)-arginine hydrochloride, Nα-tosyl-L(+)-arginine methyl hydrochloride, L(+)-arginine methyl dihydrochloride, Nα-benzoyl-L(+)-arginine ethyl hydrochloride, Nα-benzoyl-L(+)-arginine amide hydrochloride, and L(+)-arginine carbonate.

In one embodiment, human FcγRIIIa can be stabilized by adding arginine and/or its analogues at the aforementioned concentrations to a solution containing unpurified, roughly purified, or purified human FcγRIIIa. In another embodiment, when carrying out the method for producing human FcγRIIIa of the present invention described below, eluted human FcγRIIIa can be stabilized by adding arginine and/or its analogues at the aforementioned concentrations to an elution buffer. In yet another embodiment, the precipitate can be redissolved and stored by adding arginine and/or its analogues at the aforementioned concentrations to a human FcγRIIIa solution in which a precipitate has formed.

It is preferable to add glycerol to the storage solution of the present invention at a final concentration of 3 (w/v)% or more and 45 (w/v)% or less, since this further stabilizes human FcγRIIIa in the solution. It is even more preferable to add glycerol to the solution at a final concentration of 5 (w/v)% or more and 15 (w/v)% or less.

As used herein, "stabilization" means that the solubility of human FcγRIIIa is improved and precipitation is less likely to occur.

The method for producing human FcγRIIIa of the present invention includes a purification step using a column packed with a chromatography carrier. An example of a solution containing human FcγRIIIa that is applied to the column is a solution of human FcγRIIIa crudely purified from a culture medium of a host (transformant) transformed with an expression plasmid containing a polynucleotide encoding human FcγRIIIa.

Hosts for expressing human FcγRIIIa include animal cells such as COS cells and CHO (Chinese Hamster Ovary) cells, bacteria such as the genus Bacillus (including bacteria of the genus Bacillus in the broad sense, such as bacteria of the genus Brevibacillus and Paenibacillus), and Escherichia coli, yeasts such as the genus Saccharomyces, Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus. Among these, Escherichia coli is preferred as a host because it is easy to handle. When the host is Escherichia coli, human FcγRIIIa can be expressed by culturing the transformant using the methods disclosed in JP 2012-034591 A and JP 2013-085531 A, for example.

To obtain a crude human FcγRIIIa solution from the culture medium of the transformant, which is to be applied to a chromatography carrier-packed column, a suitable method may be selected depending on the form of expression. For example, when the expressed human FcγRIIIa is expressed in the periplasm of the host cells, the culture medium is centrifuged to obtain a cell-free extract containing the expressed human FcγRIIIa, which is then suspended in an appropriate buffer and disrupted (physically, chemically, etc.), followed by centrifugation to remove the disrupted residue. When the expressed human FcγRIIIa leaks from the periplasm of the host cells into the culture supernatant, the expressed human FcγRIIIa may be collected from the culture supernatant obtained by centrifuging the culture medium. When disrupting host cells with a drug, for example, disruption can be performed using the method disclosed in JP 2013-252099 A or a commercially available extraction reagent such as BugBuster Protein Extraction Reagent (Millipore).

The chromatography carrier-packed column used in the method for producing human FcγRIIIa of the present invention is not particularly limited as long as it is a column packed with a chromatography carrier that is normally used by those skilled in the art for protein purification, and examples of the carrier include a gel filtration chromatography carrier, a cation exchange chromatography carrier, an anion exchange chromatography carrier, a hydrophobic chromatography carrier, and an affinity chromatography carrier. Among these, a cation exchange chromatography carrier is preferred as a chromatography carrier in the production method of the present invention.

There are no particular limitations on the cation exchange chromatography carrier, so long as it is a carrier into which a cation exchange group such as a carboxymethyl group, a sulfopropyl group, or a sulfonic acid group has been introduced. Specific examples include TOYOPEARL CM-650, TOYOPEARL SP-650, TOYOPEARL GigaCap S-650 (all manufactured by Tosoh Corporation), and CM Sepharose Fast Flow (manufactured by Cytiva). When carrying out the purification method of the present invention using the cation exchange chromatography carrier, an appropriate open column or the like may be filled with the carrier in an amount determined by the amount of solution (apply solution) containing human FcγRIIIa added to the carrier and the protein adsorption performance of the carrier. In addition, it is advisable to equilibrate the cation exchange chromatography carrier with an appropriate buffer (Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, etc.) before adding the application solution.

A specific example of the method for purifying human FcγRIIIa of the present invention using a column packed with a carrier for cation exchange chromatography is shown below.
(I) The solution of crude human FcγRIIIa obtained by the above-mentioned method is applied to a column packed with a carrier for cation exchange chromatography that has been equilibrated in advance, and human FcγRIIIa is adsorbed onto the carrier.
(II) A washing solution containing sodium chloride is applied to the column to remove contaminating proteins.
(III) An elution buffer is applied to the column to elute human FcγRIIIa adsorbed to the carrier. The elution buffer may contain a higher sodium chloride content than the washing solution. The elution buffer may also contain arginine and/or its analog at a final concentration of 0.2 mol/L to 1 mol/L.
(IV) The fraction containing the eluted human FcγRIIIa is collected and stored. If the elution is performed with an elution buffer that does not contain arginine and/or its analog in (III), the fraction is allowed to contain arginine and/or its analog at a final concentration of 0.2 mol/L to 1 mol/L before storage.

An example of the human FcγRIIIa of the present invention is a polypeptide described in any one of (i) to (v) below.
(i) a polypeptide comprising at least the amino acid residues from glycine (Gly) at position 17 to glutamine (Gln) at position 192, which is a part of the extracellular domain (EC domain in FIG. 1 ) of the amino acid sequence of native human FcγRIIIa set forth in SEQ ID NO:1;
(ii) a polypeptide comprising at least the amino acid residues from glycine (Gly) at position 17 to glutamine (Gln) at position 192 of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions among the amino acid residues at positions 17 to 192, and having antibody-binding activity;
(iii) a polypeptide comprising at least the amino acid residues from glycine (Gly) at the 24th position to glutamine (Gln) at the 199th position of the amino acid sequence set forth in SEQ ID NO: 2;
(iv) a polypeptide having an amino acid sequence including at least the amino acid residues from glycine (Gly) at the 24th position to glutamine (Gln) at the 199th position of the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the 24th to 199th amino acid residues further include substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having antibody-binding activity;
(v) A polypeptide comprising at least the amino acid residues from glycine (Gly) at the 24th position to glutamine (Gln) at the 199th position of the amino acid sequence set forth in SEQ ID NO:2, and having a homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more to the amino acid sequence from the 24th to the 199th positions, and having antibody-binding activity.

In (ii) and (iv) above, "one or several" may mean, for example, 1 to 30, 1 to 20, or 1 to 10 (any of 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1).

Examples of (ii) include the Fc-binding protein disclosed in JP 2015-086216 A, the Fc-binding protein disclosed in JP 2016-169197 A, the Fc-binding protein disclosed in JP 2017-118871 A, the Fc-binding protein disclosed in JP 2018-197224 A, and the Fc-binding protein disclosed in WO 2019/083048.

Among these, the polypeptides described in any one of (iii) to (v) above are preferred embodiments of human FcγRIIIa in the present invention, because they tend to aggregate/precipitate in solution. The amino acid residues from glycine at position 24 to glutamine at position 199 in the amino acid sequence described in SEQ ID NO:2 described in (iii) to (v) above refer to amino acid residues from glycine at position 17 to glutamine at position 192 in the amino acid sequence described in SEQ ID NO:1, provided that the amino acid residues at positions 17 to 192 include Glu21Gly (this notation indicates that glutamic acid at position 21 in SEQ ID NO:1 is replaced with glycine, the same applies below), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51His, Gl u54Asp, Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, T The amino acid residues have the following amino acid substitutions: yr141Phe, Gly147Val, Tyr158Val, Lys165Glu, Phe171Ser, Phe176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp, and Ile190Val.

The preservation solution of the present invention may contain a salt (sodium chloride, potassium chloride, sodium sulfate, etc.) to give a final concentration of 0.5 mol/L or more, preferably 0.6 mol/L or more and 1.5 mol/L or less, preferably 1 mol/L or less, and may contain a base (sodium hydroxide, potassium hydroxide, etc.) to give a final concentration of 7.0 to 9.0, preferably 7.5 to 8.5.

The present invention is characterized by using a solution containing arginine and/or its analogs at 0.2 mol/L or more and 1 mol/L or less as a storage solution for human FcγRIIIa. The present invention improves the solubility of human FcγRIIIa and enables human FcγRIIIa to be stably stored in solution without precipitation.

A diagram showing the structure of human FcγRIIIa. FIG. 1 shows the results of measuring the saturation concentration of human FcγRIIIa in each storage solution. FIG. 1 shows the results of evaluation of storage stability of human FcγRIIIa.

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

Example 1: Examination of the composition of the storage solution (1) E. coli W3110 strain was transformed with an expression vector containing a polynucleotide encoding FcR36i_Cys consisting of the amino acid sequence set forth in SEQ ID NO: 2 to obtain a recombinant E. coli. In FcR36i_Cys (SEQ ID NO: 2), the first methionine (Met) to the 22nd alanine (Ala) are the improved PelB signal peptide (UniProt No. An oligopeptide consisting of the 1st to 22nd amino acid residues of P0C1C1, with the proviso that the 6th proline is substituted with serine), and the 24th glycine (Gly) to the 199th glutamine (Gln) are amino acid residues from the 17th to 192nd amino acid residues of human FcγRIIIa (the amino acid residues from the 17th to the 192nd amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the 17th to the 192nd amino acid residues are Glu21Gly (this notation indicates that the glutamic acid at the 21st position (the 28th position in SEQ ID NO: 2) in SEQ ID NO: 1 is substituted with glycine, the same applies below), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51H is, Glu54Asp, Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, V al117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, Tyr141Phe, Gly147Val, Tyr158V al, Lys165Glu, Phe171Ser, Phe176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp, and Ile190Val), and the cysteine tag sequence is from glycine (Gly) at position 200 to glycine (Gly) at position 207. The recombinant E. coli was cultured with reference to the method described in JP 2013-085531 A, and E. coli cells (wet cells) were obtained by centrifugation of the obtained culture solution.

(2) The wet cells obtained in (1) were suspended in 20 mmol/L phosphate buffer (pH 6.0) containing 1 mmol/L EDTA in an amount three times the weight of the wet cells, and stirred at room temperature (20°C to 25°C) until homogenous.

(3) After stirring at room temperature, Benzonase (Merck), a nuclease, was added to a final concentration of 2500 units/L, and magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol/L, and the mixture was stirred at room temperature.

(4) After stirring at room temperature, lysozyme, a carbohydrate-degrading enzyme, was added to a final concentration of 0.005 (w/v)%, and the mixture was further stirred at room temperature.

(5) After stirring at room temperature, an aqueous solution of Triton X-100 (trade name), a nonionic surfactant, was added to a final concentration of 0.5 (w/v)%, and the mixture was further stirred at room temperature.

(6) After stirring at room temperature, sodium dodecyl sulfate, an anionic surfactant, was added to a final concentration of 0.2 (w/v)%, and the mixture was further stirred at room temperature.

(7) After performing the extraction procedure overnight, the extract was centrifuged (8,000 rpm, 20 minutes, twice) to obtain the supernatant (cell-free extract).

(8) The FcR36i_Cys extract obtained in (7) was applied to a column (Merck Millipore) packed with a cation exchange chromatography carrier (TOYOPEARL CM-650M, Tosoh) that had been previously equilibrated with 20 mM phosphate buffer (pH 6.0). After washing with the buffer used for equilibration, the column was washed with 20 mmol/L phosphate buffer (pH 6.0) containing 0.4 mol/L sodium chloride to remove impurities.

(9) FcR36i_Cys was eluted with 20 mmol/L Tris-HCl buffer (pH 7.5) containing 0.7 mol/L NaCl and 10% (w/v) glycerol (hereinafter also referred to as "elution buffer A").

(10) The FcR36i_Cys solution obtained in (9) was replaced by dialysis with a 20 mmol/L Tris-HCl buffer solution (all concentrations are final concentrations) shown in any one of (a) to (o) in Table 1.

(11) The buffer-exchanged FcR36i_Cys solution was concentrated using an ultrafiltration membrane (Millipore, Amicon Ultra-0.5 mL, Ultracel-10K) until precipitation occurred. During this process, the centrifuge was frequently stopped and the solution was stirred to prevent localized high concentrations.

(12) The supernatant of the solution obtained in (11) was collected by centrifugation, and the protein concentration in the supernatant was determined from the absorbance at 280 nm. The protein concentration was determined as the saturation concentration of FcR36i_Cys for that solution.

Comparative Example 1 Evaluation of Saturation Concentration of Human FcγRIIIa in Elution Buffer (1) The same procedure as in Example 1 (10) was carried out, except that elution buffer A was used as the dialysis buffer in Example 1 (10).

The results of Example 1 and Comparative Example 1 are shown in Figure 1. Under the conditions of Table 1(i), that is, in a solution containing 0.5 mol/L L(+)-arginine (L(+)-Arg), the saturation concentration of human FcγRIIIa increased significantly. On the other hand, even when L(+)-Arg, polyethylene glycol (PEG), or ammonium sulfate (ammonium sulfate) was added at a final concentration of 0.1 mol/L or less, the saturation concentration of human FcγRIIIa was equal to or lower than that in elution buffer A (Comparative Example 1) that does not contain arginine. These results show that the inclusion of arginine and/or its analogs at a concentration of 0.2 mol/L or more and 1 mol/L or less improves the solubility of human FcγRIIIa, and allows human FcγRIIIa to be stored without precipitation.

Example 2 Evaluation of storage stability of FcR36i_Cys in arginine-containing solution (1) To the FcR36i_Cys solution obtained in Example 1 (9), L(+)-arginine and/or L(+)-arginine hydrochloride were added to give a final arginine concentration of 0.5 mol/L, and NaCl was added to give a final concentration of 0.8 mol/L. The pH was adjusted to 8.5, and the solution was mixed by inversion to obtain a homogenous solution.

(2) The arginine-containing FcR36i_Cys solution obtained in (1) was refrigerated and the protein concentration in the supernatant of the solution immediately after the start of refrigerated storage (day 0) and after 1 day, 8 days, 12 days, 22 days, and 33 days of storage was measured in the same manner as in Example 1 (12).

Comparative Example 2 Evaluation of storage stability of FcR36i_Cys in elution buffer The FcR36i_Cys solution obtained in Example 1 (9) was stored in a refrigerator, and the protein concentration in the solution supernatant immediately after the start of refrigerated storage (day 0) and after 1 day, 8 days, 12 days, 22 days, and 33 days of storage was measured in the same manner as in Example 1 (12).

The results of Example 2 and Comparative Example 2 are shown in Table 2 and Figure 3. It can be seen that the addition of arginine to a final concentration of 0.5 mol/L (Example 2) to the human FcγRIIIa storage solution significantly improved storage stability compared to when no arginine was added (Comparative Example 2). Specifically, after 33 days of refrigerated storage, the protein concentration in the storage solution that did not contain arginine (Comparative Example 2) decreased to less than one-tenth, whereas the protein concentration in the storage solution that contained 0.5 mol/L arginine (Example 2) was almost unchanged.

Claims (4)

A storage solution of human FcγRIIIa, comprising 0.5 mol/L to 1 mol/L of arginine and/or its hydrochloride ,
The storage solution, wherein human FcγRIIIa is a polypeptide selected from the group consisting of (a) to (c) below:
(a) a polypeptide comprising at least the amino acid residues from the 24th glycine to the 199th glutamine of the amino acid sequence set forth in SEQ ID NO:2;
(b) a polypeptide comprising an amino acid sequence including a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid sequence from glycine at the 24th position to glutamine at the 199th position of the amino acid sequence set forth in SEQ ID NO: 2, and having antibody-binding activity;
(c) A polypeptide comprising an amino acid sequence having 90 % or more identity to the amino acid sequence from the 24th glycine to the 199th glutamine of the amino acid sequence set forth in SEQ ID NO:2, and having antibody binding activity. The preservation solution of claim 1 , further comprising glycerol at 3% (w/v) or more and 45% (w/v) or less. A method for stabilizing human FcγRIIIa, comprising the step of contacting human FcγRIIIa with the preservation solution according to claim 1 or 2. applying a solution containing human FcγRIIIa to a column packed with a chromatography carrier to adsorb human FcγRIIIa to the carrier;
applying an elution buffer to the column to elute human FcγRIIIa adsorbed to the carrier;
recovering the eluted fraction containing human FcγRIIIa;
and storing the collected fraction,
The method for producing the composition, wherein the preserving step is a step of preserving the composition in a preservation solution according to claim 1 or 2.

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