JP7443794B2 - Stock solution of human FcγRIIIa - Google Patents

Description

The present invention relates to storage solutions of FcγRIIIa. In particular, the present invention relates to a storage solution for human FcγRIIIa that can be stored without precipitating human FcγRIIIa.

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 by recognition domains belonging to the immunoglobulin superfamily, by recognition domains on Fc receptors. This determines which accessory cells are mobilized in the immune response. Fc receptors can be further classified into several subtypes, including Fcγ receptors that 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. There is. Further, each receptor is further classified into more finely classified types, and the existence of Fcγ receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb has been reported (Non-Patent Document 1).

Among Fcγ receptors, FcγRIIIa exists on the surface of cells such as natural killer cells (NK cells) and macrophages, and is an important receptor involved in ADCC (antibody-dependent cytotoxicity) activity, which is important in the human immune system. It is. It has been reported that the affinity between FcγRIIIa and human IgG has a K _D value, which indicates the strength of binding, of 10 ⁻⁷ M or less (Non-Patent Document 2). Furthermore, it is known that the binding properties between FcγRIIIa and antibodies differ due to differences in the sugar chain structures of the antibodies (Non-Patent Document 3).

The amino acid sequence of natural human FcγRIIIa (SEQ ID NO: 1) has been published in public databases such as UniProt (Accession number: P08637). Furthermore, the structural functional domains of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the location of the cell membrane-penetrating region have also been published. FIG. 1 shows a schematic structural diagram of human FcγRIIIa. Note that 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, from the 1st methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1 is the signal sequence (S), and from the 17th glycine (Gly) to the 208th glutamine (Gln) is the extracellular region. (EC), the 209th valine (Val) to the 229th valine (Val) is the transmembrane region (TM), and the 230th lysine (Lys) to the 254th lysine (Lys) is the intracellular region (C). ). It is known that FcγRIIIa binds particularly strongly to IgG1 and IgG3 among human IgG subclasses from IgG1 to IgG4, but weakly binds to IgG2 and IgG4.

Since human FcγRIIIa recognizes the sugar chain structure of an antibody, an adsorbent obtained by immobilizing human FcγRIIIa on an insoluble carrier can separate antibodies based on the difference in binding properties based on the sugar chain structure (Patent Documents 1 and 2). . Since the sugar chain structure of an antibody has a large influence on the efficacy and stability of antibody drugs, it is extremely important to control the sugar chain structure when producing antibody drugs. Therefore, the adsorbent is useful for process analysis and fractionation during antibody drug production.

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

JP2015-086216A Japanese Patent Application Publication No. 2016-169197 JP 2016-183113 Publication

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

An object of the present invention is to provide a stabilization method and a storage solution for human FcγRIIIa that can stably store human FcγRIIIa (particularly human FcγRIIIa with improved alkaline resistance through amino acid substitution) without precipitation.

As a result of intensive studies to solve the above problem, we found that human FcγRIIIa does not precipitate when it contains glycerol at a final concentration of 3% (w/v) to 45% (w/v). This led to the completion of the present invention.

That is, the present invention includes the embodiments described in [1] to [4] below.

[1]
A stock solution of human FcγRIIIa, the stock solution comprising 3% (w/v) to 45% (w/v) glycerol.

[2]
A method for stabilizing human FcγRIIIa, the method comprising contacting human FcγRIIIa with 3% (w/v) to 45% (w/v) glycerol.

[3]
Applying a solution containing human FcγRIIIa to a column packed with a chromatography carrier, and adsorbing human FcγRIIIa to the carrier;
applying an elution buffer to the column and eluting human FcγRIIIa adsorbed to the carrier;
Collecting the eluted fraction containing human FcγRIIIa;
A method for producing human FcγRIIIa, comprising a step of preserving the collected fraction,
The manufacturing method described above, wherein the storing step is performed using the storage solution according to [1].

[4]
The storage solution according to [1], wherein the human FcγRIIIa is a polypeptide according to any one of the following (a) to (c).
(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) Contains 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; A polypeptide having an amino acid sequence containing a substitution, deletion, insertion, or addition of one or several amino acid residues at a position and having antibody binding activity;
(c) Contains 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, provided that it has 80% or more homology to the amino acid sequence from the 24th to the 199th. and has antibody binding activity.

The present invention will be explained in detail below.

The stock solution of human FcγRIIIa of the present invention is characterized in that the solution containing human FcγRIIIa contains glycerol at a final concentration of 3% (w/v) to 45% (w/v), and glycerol is present in the solution. Precipitation of human FcγRIIIa can be suppressed. The final concentration of glycerol contained in the human FcγRIIIa storage solution is preferably in the range of 5% (w/v) to 40% (w/v).

In one embodiment, human FcγRIIIa can be stabilized by adding glycerol at the concentration described above 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 glycerol at the concentration described above to the elution buffer.

Here, "stabilization" means that precipitation of human FcγRIIIa is less likely to occur.

The method for producing human FcγRIIIa of the present invention includes a purification step using a carrier-packed column for chromatography. An example of a solution containing human FcγRIIIa to be applied to the column is a human FcγRIIIa solution roughly purified from a culture solution 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 cells, Bacillus (including Bacillus in a broad sense such as Brevibacillus and Paenibacillus), and Escherichia coli. Bacteria such as Saccharomyces, Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus oryzae can be used. Among these, it is preferable to use Escherichia coli as the host because it is easy to handle. In addition, when the host is E. coli, human FcγRIIIa may be expressed by culturing a transformant by the methods disclosed in JP-A No. 2012-034591 and JP-A No. 2013-085531.

In order to obtain a crudely purified human FcγRIIIa solution to be applied to a column packed with a carrier for chromatography from the culture solution of the transformant, an appropriate selection may be made depending on the form of expression. For example, when the expressed human FcγRIIIa is expressed in the periplasm of host cells, the host cells obtained by centrifuging the culture solution are suspended in an appropriate buffer, and after cell disruption (physical disruption, disruption with drugs, etc.), It is sufficient to obtain a cell-free extract containing the expressed human FcγRIIIa by removing the fragmentation residue by centrifugation. If the expressed human FcγRIIIa leaks from the periplasm of the host cell into the culture supernatant, the culture solution should be centrifuged. The expressed human FcγRIIIa may be recovered from the culture supernatant obtained by separation. Note that when disrupting host cells with a drug, for example, the method disclosed in JP-A-2013-252099 or a commercially available extraction reagent such as BugBuster Protein extraction kit (manufactured by Takara Bio Inc.) may be used.

The column packed with a carrier for chromatography 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 carrier for chromatography commonly used in protein purification by those skilled in the art. , carriers for cation exchange chromatography, carriers for anion chromatography, carriers for hydrophobic chromatography, and carriers for affinity chromatography. Among these, cation exchange chromatography carriers are preferred as chromatography carriers in the production method of the present invention.

The carrier for cation exchange chromatography is not particularly limited as long as it has a cation exchange group such as a carboxymethyl group, sulfopropyl group, or sulfonic acid group introduced into the carrier, and specific examples include TOYOPEARL CM-650 and TOYOPEARL SP-. 650, TOYOPEARL GigaCap S-650 (manufactured by Tosoh), and CM Sepharose Fast Flow (manufactured by GE Healthcare). When carrying out the purification method of the present invention using the carrier for cation exchange chromatography, the amount of the solution containing human FcγRIIIa (apply solution) added to the carrier, the protein adsorption performance of the carrier, etc. The determined amount of carrier may be packed into an appropriate open column or the like. Further, the carrier for cation exchange chromatography is preferably equilibrated in advance with an appropriate buffer solution (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 carrier-packed column for cation exchange chromatography is shown below.
(I) The crudely purified human FcγRIIIa solution obtained by the method described above 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 contaminant proteins.
(III) Applying an elution buffer to the column to elute human FcγRIIIa adsorbed to the carrier. As the elution buffer, a buffer containing higher sodium chloride than the washing solution may be used. The elution buffer may also contain glycerol at a final concentration of 3% (w/v) to 45% (w/v).
(IV) The eluted fraction containing human FcγRIIIa is collected and stored. In addition, when elution is performed with an elution buffer that does not contain glycerol in (III), the fraction is stored after containing glycerol at a final concentration of 3% (w/v) to 45% (w/v).

An example of human FcγRIIIa in the present invention includes a polypeptide described in any one of (i) to (v) below.
(i) Amino acid residues from glycine at position 17 to glutamine at position 192, which is part of the extracellular region (EC region in Fig. 1), of the amino acid sequence of natural human FcγRIIIa described in SEQ ID NO: 1. A polypeptide comprising at least;
(ii) Contains at least the amino acid residues from the 17th glycine to the 192nd glutamine of the amino acid sequence set forth in SEQ ID NO: 1, provided that one or several amino acid residues from the 17th to 192nd amino acid residues are included. A polypeptide that further has any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at a position, and has antibody binding activity;
(iii) 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;
(iv) Contains 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, provided that one or more amino acid residues are further contained in the 24th to 199th amino acid residues A polypeptide having an amino acid sequence containing a substitution, deletion, insertion, or addition of one or several amino acid residues at a position and having antibody binding activity;
(v) Contains 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, more preferably 80% or more of the amino acid sequence from the 24th to the 199th. has a homology of 90% or more, more preferably 95% or more, and has antibody binding activity.

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

As an example of the above (ii), the Fc binding protein disclosed in JP2015-086216A, the Fc binding protein disclosed in JP2016-169197A, the Fc binding protein disclosed in JP2017-118871A, Fc-binding proteins disclosed in JP-A-2018-197224, and Fc-binding proteins disclosed in WO2019/083048.

Among them, the polypeptide described in any one of (iii) to (v) above is a preferred embodiment of human FcγRIIIa in the present invention because it easily aggregates/precipitates in a solution. Note that the amino acid residues from the 24th glycine to the 199th glutamine of the amino acid sequence set forth in SEQ ID NO: 2, described in (iii) to (v) above, refer to the 17th amino acid residue of the amino acid sequence set forth in SEQ ID NO: 1. The amino acid residues from the glycine to the 192nd glutamine are the amino acid residues from the 17th to the 192nd amino acid residues. The same applies hereinafter), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51His, Glu54Asp, Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Ph e, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, Tyr141Phe, Gly147Val, Tyr158Val, Lys165Glu, Phe171Ser, Val176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn 187Asp and Ile190Val amino acid residues.

Note that the storage solution of the present invention contains a salt (sodium chloride, potassium chloride, sodium sulfate, etc.) so that the final concentration is 0.5M or more, preferably 0.8M or more, 1.5M or less, preferably 1.0M or less. A base (sodium hydroxide, potassium hydroxide, etc.) may be included to adjust the pH to 7.0 to 8.0, preferably around 7.5.

The present invention is characterized in that a solution containing 3% (w/v) to 45% (w/v) glycerol is used as a storage solution for human FcγRIIIa. According to the present invention, human FcγRIIIa can be stably stored in a solution without precipitation.

A diagram showing the structure of human FcγRIIIa.

EXAMPLES Hereinafter, the present invention will be explained in more detail using examples, but the present invention is not limited to the above examples.

Example 1 Production of human FcγRIIIa (FcR36i_Cys) (Part 1)
(1) A transformant expressing FcR36i_Cys (SEQ ID NO: 2) prepared by the method described in WO2019/083048 was transferred to a 400 mL 2YT liquid medium containing 100 μg/mL carbenicillin (16 g peptone) in a 2 L baffled flask. /L, yeast extract 10g/L, sodium chloride 5g/L) and precultured by aerobically shaking culture at 37°C overnight. In addition, in FcR36i_Cys (SEQ ID NO: 2), from the 1st methionine (Met) to the 22nd alanine (Ala) is an oligopeptide consisting of the amino acid residues from the 1st to 22nd of the improved PelB signal peptide (UniProt No. P0C1C1). However, it is an oligopeptide in which the 6th proline is replaced with serine), and the amino acid sequence from the 24th glycine (Gly) to the 199th glutamine (Gln) is the same as the amino acid sequence of human FcγRIIIa (SEQ ID NO: 1). Amino acid residues from the 17th to the 192nd in the amino acid sequence, however, in the amino acid residues from the 17th to the 192nd, Glu21Gly, Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51His, Glu54Asp , Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, T hr140Met, Tyr141Phe, Gly147Val, Tyr158Val, Lys165Glu, Phe171Ser, Val176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp and The amino acid sequence of the polypeptide having the amino acid substitution of Ile190Val) is the cysteine tag sequence from the 200th glycine (Gly) to the 207th glycine (Gly).

(2) Glucose 10g/L, yeast extract 20g/L, trisodium phosphate dodecahydrate 3g/L, disodium hydrogen phosphate dodecahydrate 9g/L, ammonium chloride 1g/L and carbenicillin 100mg/L 180 mL of the culture solution obtained in (1) was inoculated into 1.8 L of a liquid medium containing L, and main culture was performed using a 3 L fermentor (manufactured by Biot). Main culture was started under the following conditions: temperature 30° C., pH 6.9 to 7.1, aeration rate 1 VVM, and dissolved oxygen concentration 30% saturation. To control the pH, 50% phosphoric acid was used as the acid, and 14% ammonia water was used as the alkali.Dissolved oxygen was controlled by changing the stirring speed, and the stirring rotation speed was set to a lower limit of 500 rpm and an upper limit of 1000 rpm. . After the start of culture, when the glucose concentration can no longer be measured, the fed-batch medium (glucose 248.9 g/L, yeast extract 83.3 g/L, magnesium sulfate heptahydrate 7.2 g/L) is added to dissolved oxygen (DO). ) was added under control.

(3) When the absorbance at 600nm (OD600nm) reaches approximately 150 as a guideline for the amount of bacterial cells, lower the culture temperature to 25℃, and after confirming that the set temperature has been reached, adjust the final concentration to 0.5mM. IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added, and the culture was continued at 25°C.

(4) About 48 hours after the start of the culture, the culture was stopped, and the culture solution was centrifuged at 4° C. and 8000 rpm for 20 minutes to collect the bacterial cells.

(5) After collecting cells from the recombinant E. coli culture solution, extract solution (1mM EDTA, 40mM sodium chloride, 2mM magnesium sulfate, 250 Unit/L Benzonase (manufactured by Merck), 0.005% (w/ Extract the protein expressed within the bacterial cells using 20 mM phosphate buffer (pH 6.0) containing lysozyme (v) and 0.5% (w/v) Triton X-100 (trade name). The bacterial cell extract was centrifuged twice at 15,000 rpm for 20 minutes at 4°C to obtain an FcR36i_Cys extract.

(6) The FcR36i_Cys extract obtained in (5) was packed into 140 mL of a cation exchange chromatography carrier (TOYOPEARL CM-650M, manufactured by Tosoh) that had been equilibrated in advance with 20 mM phosphate buffer (pH 6.0). It was applied to a VL32×250 column (manufactured by Merck Millipore) at a flow rate of 13 mL/min. After washing with the buffer used for equilibration, contaminant impurities were removed by washing with 20 mM phosphate buffer (pH 6.0) containing 0.4 M sodium chloride.

(7) FcR36i_Cys was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing 0.7 M sodium chloride. After elution, FcR36i_Cys was diluted with 20 mM Tris-HCl buffer (pH 7.5) containing 0.7 M sodium chloride so that the concentration was 1 mg/mL.

(8) To the FcR36i_Cys solution obtained in (7) with a concentration of 1 mg/mL, add sodium chloride so that the salt concentration is 0.8 M or more, and add sodium hydroxide aqueous solution so that the pH becomes 8.5. Glycerol was added to each sample at a final concentration of 5% (w/v) and stored at 4°C for 4 days.

(9) The presence or absence of precipitation in the FcR36i_Cys solution after storage in (8) was visually confirmed.

Example 2 Production of FcR36i_Cys (Part 2)
FcR36i_Cys was purified in the same manner as in Example 1, except that the final concentration of glycerol added in Example 1 (8) was 20% (w/v), and after storage, the presence or absence of precipitation was confirmed. .

Example 3 Production of FcR36i_Cys (Part 3)
FcR36i_Cys was purified in the same manner as in Example 1, except that the final concentration of glycerol added in Example 1 (8) was 40% (w/v), and after storage, the presence or absence of precipitation was confirmed. .

Example 4 Production of FcR36i_Cys (Part 4)
(1) An FcR36i_Cys extract was obtained in the same manner as in Example 1 (1) to (5).

(2) The FcR36i_Cys extract was applied to the column in the same manner as in Example 1 (6), and contaminant impurities were removed by washing.

(3) Same as Example 1 (7) except that 20 mM Tris-HCl buffer (pH 7.5) containing 0.7 M sodium chloride and 10% (w/v) glycerol was used as the elution buffer. FcR36i_Cys was eluted using the method described above.

(4) To the FcR36i_Cys solution obtained in (3) with a concentration of 1 mg/mL, add sodium chloride so that the salt concentration is 0.8 M or more, and add sodium hydroxide aqueous solution so that the pH becomes 8.5. Each was added and stored at 4°C for 4 days. Note that the addition of the sodium chloride and sodium hydroxide aqueous solutions has only a slight effect on the glycerol concentration in the FcR36i_Cys solution.

(5) The presence or absence of precipitate was confirmed in the same manner as in Example 1 (9).

Comparative Example 1 Production of FcR36i_Cys (Part 5)
FcR36i_Cys was purified in the same manner as in Example 1 except that glycerol was not added in Example 1 (8), and after storage, the presence or absence of precipitation was confirmed.

Comparative Example 2 Production of FcR36i_Cys (Part 6)
FcR36i_Cys was purified in the same manner as in Example 1, except that the final concentration of glycerol added in Example 1 (8) was 50% (w/v), and after storage, the presence or absence of precipitation was confirmed. .

Comparative Example 3 Production of FcR36i_Cys (Part 7)
FcR36i_Cys was purified in the same manner as in Example 1, except that sucrose was added in place of glycerol in Example 1 (8) to a final concentration of 1000 mM, and after storage, the presence or absence of precipitation was confirmed.

Comparative Example 4 Production of FcR36i_Cys (Part 8)
FcR36i_Cys was purified in the same manner as in Example 1, except that D-sorbitol was added to a final concentration of 800 mM instead of glycerol in Example 1 (8), and after storage, the presence or absence of precipitation was confirmed. did.

Comparative Example 5 Production of FcR36i_Cys (Part 9)
FcR36i_Cys was added in the same manner as in Example 1, except that Tween 20 (trade name) was added in place of glycerol in Example 1 (8) to a final concentration of 0.01% (w/v). After purification and storage, the presence or absence of precipitate was confirmed.

Table 1 shows the results of confirming the presence or absence of precipitate. When glycerol was added and stored (Examples 1 to 4 and Comparative Example 2), human FcγRIIIa in solution was stabilized and no precipitation occurred. On the other hand, when the solution was stored without addition (Comparative Example 1) or with the addition of saccharides other than glycerol (Comparative Examples 3 and 4) or surfactant (Comparative Example 5), human FcγRIIIa in the solution aggregated and precipitated.

Example 5 Measurement of antibody adsorption amount by human FcγRIIIa immobilized carrier (1) After activating the hydroxyl groups present on the surface of porous hydrophilic polymer particles with a particle size of 10 μm with iodoacetyl groups, the activated carrier was injected from Example 1. By reacting FcR36i_Cys produced in Example 4 and Comparative Example 2, an FcR36i-immobilized carrier was obtained.

(2) PBS (Phosphate Buffered Saline) (pH 7.4) was added to 1 mL of the immobilization carrier prepared in (1) to make a 50% slurry.

(3) After homogenizing the prepared slurry, 100 μL of the slurry (50 μL as immobilization carrier) was added to a spin column (Cosmos Spin Filter H 0.45 μm, manufactured by Nacalai Tesque) and centrifuged at 3000 rpm for 1 minute. , a suction-dried immobilization carrier was prepared.

(4) 150 μL of PBS was added to the suction-dried immobilization carrier and centrifuged at 3000 rpm for 1 minute. The immobilization carrier was washed by repeating this operation three times.

(5) Sequentially add 150 μL of PBS and 60 μL of human immunoglobulin (globulin intramuscular injection 1500 mg/10 mL “JB”, manufactured by Japan Blood Products Organization) to the immobilization carrier after washing, and stir at 25°C for 2 hours. , immunoglobulin (antibody) was adsorbed onto the immobilization carrier.

(6) After the adsorption operation in (5), the gel was separated from the solution containing unadsorbed antibodies by centrifuging the spin column at 3000 rpm for 1 minute.

(7) 150 μL of PBS was added to the immobilization carrier and centrifuged at 3000 rpm for 1 minute. The immobilization carrier was washed by repeating this operation three times.

(8) 150 μL of 50 mM citrate buffer (pH 3.0) was added to the gel, and centrifuged at 3000 rpm for 1 minute. By repeating this operation three times, the antibody adsorbed to the immobilization carrier was eluted. The concentration of the antibody was calculated by measuring the absorbance of the eluate, and the amount of antibody adsorbed onto the FcR36i immobilized carrier was determined.

The results are shown in Table 2. When glycerol with a final concentration of 5% (w/v) to 40% (w/v) was used as the human FcγRIIIa storage solution (Examples 1 to 4), the amount of antibody adsorbed on the human FcγRIIIa-immobilized carrier was It can be seen that the antibody binding ability of the stored human FcγRIIIa is maintained at 10 mg per gram of carrier. On the other hand, when glycerol with a final concentration of 50% (w/v) was used as a storage solution for human FcγRIIIa (Comparative Example 2), the amount of antibody adsorbed on the carrier immobilized with human FcγRIIIa was less than 1 mg per 1 g of carrier, and the storage solution The antibody binding ability of human FcγRIIIa was significantly reduced. The above results indicate that the final concentration of glycerol added as a human FcγRIIIa storage solution is preferably from 3% (w/v) to 45% (w/v).

Claims (3)

A storage solution for human FcγRIIIa, the storage solution comprising 3% (w/v) to 45% (w/v) glycerol, the storage solution comprising:
The storage solution, wherein the human FcγRIIIa is a polypeptide according to any one 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) Contains 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; A polypeptide having an amino acid sequence containing substitution, deletion, insertion, or addition of amino acid residues and having antibody binding activity;
(c) Contains 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, provided that it has 90% or more identity with the amino acid sequence from the 24th to the 199th. and has antibody binding activity. A method for stabilizing human FcγRIIIa, the method comprising contacting human FcγRIIIa with 3% (w/v) to 45% (w/v) glycerol, the method comprising:
The stabilization method, wherein the human FcγRIIIa is a polypeptide according to any one 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) Contains 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; A polypeptide having an amino acid sequence containing substitution, deletion, insertion, or addition of amino acid residues and having antibody binding activity;
(c) Contains 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, provided that it has 90% or more identity with the amino acid sequence from the 24th to the 199th. and has antibody binding activity. Human FcγRIIIa is a polypeptide according to any one of the following (a) to (c),
A step of applying a solution containing the human FcγRIIIa to a column packed with a carrier for chromatography and adsorbing human FcγRIIIa to the carrier; a step of applying an elution buffer to the column and eluting the human FcγRIIIa adsorbed to the carrier;
Collecting the eluted fraction containing human FcγRIIIa;
A method for producing human FcγRIIIa, comprising a step of preserving the collected fraction,
The said manufacturing method which preserve|saves with the preservation |save solution of Claim 1 in the preservation|save process.
(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) Contains 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; A polypeptide having an amino acid sequence containing substitution, deletion, insertion, or addition of amino acid residues and having antibody binding activity;
(c) Contains 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, provided that it has 90% or more identity with the amino acid sequence from the 24th to the 199th. and has antibody binding activity.