JP7639440B2 - Antibody adsorbent with immobilized Fc receptor - Google Patents

Description

The present invention relates to an antibody adsorbent having an immobilized Fc receptor. More specifically, the present invention relates to the adsorbent having an improved antibody adsorption capacity compared to conventional adsorbents.

Antibody drugs are medicines that use antibodies (immunoglobulins), molecules that are responsible for the immune system in the body. Due to the diversity of the variable regions of antibodies, antibody drugs bind to target molecules with high specificity and affinity. As a result, antibody drugs have few side effects, and in recent years, the range of diseases for which they can be used has been expanding, leading to a rapid expansion of the market.

It is known that the sugar chain structure of an antibody drug is greatly related to its efficacy and stability. Therefore, when manufacturing an antibody drug, it is extremely important to control the sugar chain structure. Among antibody-binding proteins, FcγRIIIa, which is an Fc receptor, is known to recognize the sugar chain structure of an antibody (immunoglobulin), and by using an adsorbent in which FcγRIIIa is immobilized on an insoluble carrier as an affinity ligand, the antibody can be separated based on the sugar chain structure (Patent Document 1). Therefore, the adsorbent is useful for process analysis during the manufacture of antibody drugs. In addition, by creating a mutant in which an amino acid residue at a specific position in the extracellular region of FcγRIIIa (specifically, the region from the 17th glycine to the 192nd glutamine in the amino acid sequence described in SEQ ID NO: 1) is replaced with another specific amino acid residue, the thermal stability, acid stability, and alkaline stability required for an affinity ligand are improved (Patent Documents 1 to 3).

However, when attempts were made to apply an antibody adsorbent comprising an insoluble carrier and FcγRIIIa or the variant immobilized on the carrier to separate antibodies based on their glycan structures, the amount of antibody adsorbed was insufficient, making it difficult to apply the adsorbent to separate antibodies in the industrial production of antibody pharmaceuticals.

Patent Document 4 discloses that the amount of antibody adsorption is improved by immobilizing an antibody-binding protein (Protein A derived from Staphylococcus aureus) on an insoluble carrier via a peptide linker having a length of 0.9 to 91 nm and containing polyproline. On the other hand, there has been insufficient research into the use of peptide linkers to improve the amount of adsorption in antibody adsorbents with immobilized Fc receptors.

JP 2015-086216 A JP 2016-169197 A JP 2017-118871 A WO2017/018437

The objective of the present invention is to provide an antibody adsorbent with immobilized Fc receptors that can adsorb more antibodies than conventional adsorbents and can be used for antibody separation.

As a result of extensive research to solve the above problems, we were able to obtain an antibody adsorbent with improved antibody adsorption capacity compared to conventional methods by optimizing the length of the peptide linker for immobilizing the Fc receptor on the insoluble carrier.

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

[1] An antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier, the peptide linker having a length of 2 nm or more.

[2] The adsorbent according to [1], wherein the peptide linker is a polypeptide shown in either (1) or (2) below:
(1) A polypeptide comprising at least the amino acid residues from the 24th proline to the 105th histidine in the amino acid sequence set forth in SEQ ID NO:1;
(2) A polypeptide having an amino acid sequence comprising at least the amino acid residues from the 24th proline to the 105th histidine of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that the 24th to 105th amino acid residues further include substitution, deletion, insertion or addition of one or several amino acid residues at one or several positions.

[3] The adsorbent according to [2], wherein the peptide linker is a polypeptide containing at least the amino acid residues from the 6th proline to the 87th histidine in the amino acid sequence set forth in SEQ ID NO:17.

[4] The adsorbent according to any one of [1] to [3], wherein the Fc receptor is a polypeptide shown in either (i) or (ii) below:
(i) a polypeptide comprising at least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1;
(ii) A polypeptide having an amino acid sequence comprising at least the amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that the amino acid sequence further includes substitution, deletion, insertion or addition of one or more amino acid residues at one or more positions within the amino acid residues at positions 17 to 192, and having binding activity to an antibody.

[5] A method for purifying an antibody, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent according to any one of [1] to [4] to adsorb the antibody to the adsorbent; and eluting the antibody adsorbed to the adsorbent with an elution solution.

The present invention is described in detail below.

In the present invention, the Fc receptor immobilized on an insoluble carrier is a protein that has binding affinity to the Fc region of an antibody (immunoglobulin). Specific examples of human Fc receptors include human FcγRI, human FcγRIIa, human FcγRIIb, human FcγRIIIa, and human FcRn. In particular, human FcγRIIIa is a human Fc receptor that can recognize the sugar chain structure of an antibody, and an antibody separation agent in which human FcγRIIIa is immobilized on an insoluble carrier can separate antibodies based on their sugar chain structure (JP Patent Publication No. 2015-086216), and therefore this is a preferred embodiment of the Fc receptor immobilized on an insoluble carrier in the present invention.

A preferred embodiment of human FcγRIIIa is
(i) a polypeptide comprising at least the extracellular region of native human FcγRIIIa (amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence set forth in SEQ ID NO:1), and (ii) a polypeptide comprising at least the extracellular region of native human FcγRIIIa, 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 constituting the extracellular region, and having antibody-binding activity;
In addition, a more preferred embodiment of the above (ii) is as follows:
Fc-binding proteins disclosed in JP2015-086216A;
Fc-binding proteins disclosed in JP2016-169197A;
Fc binding proteins disclosed in JP2017-118871A;
Fc-binding proteins disclosed in JP2018-197224A;
Fc binding proteins as disclosed in WO2019/083048;
Examples include:

In the present invention, the insoluble carrier on which the Fc receptor is immobilized may be any material that has an activated functional group capable of immobilizing a peptide linker described below and is insoluble in a solution or solvent used for adsorption/elution of the antibody. The carrier may be an inorganic material such as zirconia, zeolite, silica, or coated silica, a natural organic polymer material such as cellulose, agarose, or dextran, or a synthetic organic polymer material such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, or vinyl polymer. The activated functional group may be bonded to a known chromatography carrier such as Shodex (Showa Denko K.K.), Sepharose (Cytiva Corporation), Amberlite (DuPont), Cellufine (JNC Corporation), POROS (Thermo Fisher Scientific), or Toyopearl (Tosoh Corporation).

The adsorbent of the present invention is characterized in that the length of the peptide linker for immobilizing the above-mentioned Fc receptor on the above-mentioned insoluble carrier is 2 nm or more. The length of the peptide linker is preferably 2 nm or more and 10 nm or less, and more preferably 3 nm or more and 8 nm or less. The sequence of the polypeptide constituting the peptide linker is not particularly limited as long as it satisfies the above-mentioned length condition. Examples include a sequence in which Pro (proline)-Ala (alanine) is repeated 8 times or more, a sequence in which Gly (glycine)-Gly-Ser (serine) is repeated 5 times or more, and a sequence in which Glu (glutamic acid)-Ala (alanine)-Ala-Ala-Lys (lysine) (SEQ ID NO: 2) is repeated 3 times or more.

A partial sequence of an Fc receptor may be used as the peptide linker. A preferred embodiment of a partial sequence of an Fc receptor as a peptide linker is a polypeptide comprising the D1 domain of human FcγRIIIa. Specifically,
(1) A polypeptide comprising at least the amino acid residues from the 24th proline to the 105th histidine in the amino acid sequence set forth in SEQ ID NO: 1, or (2) a polypeptide having an amino acid sequence comprising at least the amino acid residues from the 24th proline to the 105th histidine in the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the 24th to 105th amino acid residues further include substitution, deletion, insertion or addition of one or several amino acid residues at one or several positions.
A preferred embodiment of the above (2) is a polypeptide containing at least the amino acid residues from the 6th proline to the 87th histidine in the amino acid sequence of SEQ ID NO:17.

An oligopeptide for immobilization on an insoluble carrier may be further added to the C-terminus of the aforementioned peptide linker. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and a cysteine tag consisting of the amino acid sequence set forth in SEQ ID NO: 3.

To produce the adsorbent of the present invention, a polypeptide having the above-mentioned peptide linker added to the C-terminus of an Fc receptor is prepared, and then the polypeptide is immobilized on an insoluble carrier to produce the adsorbent. When adding a peptide linker to an Fc receptor, a polynucleotide encoding the peptide linker may be prepared, and then the peptide linker may be added to the C-terminus of the Fc receptor by genetic engineering using a method well known to those skilled in the art, or a chemically synthesized peptide linker may be chemically bonded to the C-terminus of the Fc receptor of the present invention to add the peptide linker. Furthermore, a signal peptide may be added to the N-terminus of the Fc receptor to promote efficient expression in a host. Examples of the signal peptide when the host is E. coli include signal peptides that secrete proteins into the periplasm, such as PelB (oligopeptide consisting of the 1st to 22nd amino acid residues of UniProt No. P0C1C1), DsbA, MalE (oligopeptide consisting of the 1st to 26th amino acid residues of UniProt No. P0AEX9), and TorT (JP Patent Publication No. 2011-097898). In addition, the DsbC signal peptide (the region from the 1st to 21st amino acid residues of UniProt No. P0AEG7) and oligopeptides in which one or several amino acid residues other than the N-terminus (the 1st methionine) and the C-terminus (the 20th alanine and the 21st aspartic acid) of the signal peptide have been deleted may be added to the N-terminus of the ligand.

To immobilize an Fc receptor with a peptide linker attached to an insoluble carrier, an active group such as an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetamide group may be added to the insoluble carrier, and the peptide linker may be covalently bonded to the insoluble carrier via the active group to immobilize the insoluble carrier. A carrier with an active group may be prepared, for example, by introducing the active group onto the carrier surface under appropriate reaction conditions.

On the other hand, an example of a method for introducing active groups onto the carrier surface is to react one of the compounds having two or more active sites with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc. present on the carrier surface. Examples of such compounds that introduce epoxy groups into hydroxyl groups or amino groups on the carrier surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. After introducing an epoxy group onto the carrier surface using the above compound, examples of the compound for introducing a carboxy group onto the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, and 4-aminobutyric acid. Examples of the compound for introducing a maleimide group onto a hydroxy group, epoxy group, carboxy group, or amino group present on the carrier surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-[4-N-maleimidophenyl]acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, and 1-(4-aminophenyl)maleimide. Examples include -(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the support surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester. Another example of a method is to react hydroxyl or amino groups present on the support surface with an ω-alkenylalkane glycidyl ether, and then activate the ω-alkenyl moiety by halogenating it with a halogenating agent. Examples of ω-alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

Another example of a method for introducing active groups onto the carrier surface is to introduce active groups onto carboxy groups present on the carrier surface using a condensation agent and an additive. Condensation agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

Buffers used when immobilizing Fc receptors on an insoluble carrier include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization may be appropriately set within the range of 5°C to 50°C, taking into consideration the reactivity of the active group and the stability of the Fc receptor, and is preferably in the range of 10°C to 35°C.

To separate antibodies using the antibody adsorbent of the present invention obtained by immobilizing Fc receptors on an insoluble carrier using the method described above, an equilibration solution is added to a column packed with the antibody adsorbent using a liquid delivery means such as a pump to equilibrate the column, and an antibody-containing solution is added using the liquid delivery means to specifically adsorb the antibody to the antibody adsorbent, after which an appropriate elution solution is added using the liquid delivery means to elute the adsorbed antibody. Note that in the present invention, antibody separation is not limited to antibody separation from a solution containing impurities (removal of impurities), but also includes separation between antibodies based on structure, properties, activity, etc.

Examples of the equilibration solution include phosphate buffer, acetate buffer, MES buffer, and citrate buffer. The buffer may further contain 10 mM to 100 mM (preferably 40 mM to 60 mM) of an inorganic salt such as sodium chloride. The pH of the equilibration solution may be appropriately determined from a range of pH 4.0 to 7.0, taking into consideration the buffer components, column shape, and the packing pressure of the adsorbent into the column.

To elute the antibody adsorbed to the antibody adsorbent, the interaction between the antibody and the Fc receptor may be weakened, and specific examples include lowering the pH with a buffer, adding a counter peptide, increasing the temperature, and changing the salt concentration. Specific examples of elution solutions for eluting the antibody adsorbed to the antibody adsorbent include buffer solutions that are more acidic than the solution used to adsorb the antibody to the antibody adsorbent. Examples of such buffer solutions include citrate buffer, glycine hydrochloride buffer, and acetate buffer, which have buffering capacity on the acidic side. The pH of the elution solution may be set within a range that does not impair the functions of the antibody (such as binding to an antigen), and examples include pH 2.5 to 6.0, pH 3.0 to 5.0, and pH 3.0 to 4.0.

When the antibody is eluted by changing the salt concentration, it may be eluted in one step with a buffer solution (eluent) containing a high concentration of salt, the salt concentration may be increased stepwise at will (step gradient), or the salt concentration may be increased with a linear concentration gradient (linear gradient), but elution with a linear gradient is preferred. For example, when sodium chloride is used as the water-soluble salt, elution may be performed with a linear gradient of sodium chloride concentration from 0M to 1M. When the antibody is eluted by changing the pH, it may be eluted in one step with an acidic buffer solution (eluent) with a lower pH than the equilibration buffer, the pH of the buffer may be decreased stepwise at will (step gradient), or the pH of the buffer may be decreased with a linear concentration gradient (linear gradient), but elution with a linear gradient is preferred. For example, elution may be performed with a linear gradient from a neutral to weakly acidic buffer solution to which the antibody is adsorbed to an acidic buffer solution to which the antibody is eluted.

The antibody can be obtained by collecting the antibody-containing fraction eluted by the above-mentioned method. Collection may be performed by a conventional method. Specific examples include a method of replacing the collection container at regular intervals or at regular volumes, a method of changing the collection container according to the shape of the chromatogram of the eluate, and a method of collecting fractions using an automatic fraction collector, etc.

The present invention is characterized in that, in an antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier, the length of the peptide linker is 2 nm or more. The adsorbent of the present invention has an increased antibody adsorption amount compared to a conventional adsorbent having an immobilized Fc receptor that does not have the peptide linker. Therefore, the present invention is useful for separating antibodies in the industrial production of antibody pharmaceuticals.

In particular, when human FcγRIIIa is used as the Fc receptor, the antibody separation agent obtained by immobilizing these proteins on an insoluble carrier can perform separation based on glycan structure (JP Patent Publication No. 2015-086216) or on the strength of antibody-dependent cellular cytotoxicity (JP Patent Publication No. 2016-023152), making it possible to selectively and mass-produce antibodies with specific glycan structures or antibodies with high (or low) antibody-dependent cellular cytotoxicity.

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

Example 1 Preparation of an insoluble carrier (1) Introduction of epoxy groups into a gel having hydroxyl groups (1-1) A slurry of a polymethacrylate gel (Toyopearl, manufactured by Tosoh Corporation) having hydroxyl groups on its surface was filtered by suction on a glass filter, and then dried by suction to prepare a suction-dried gel.
(1-2) 5 mL of water, 0.5 mL of 0.2 M aqueous sodium hydroxide solution, and 5 mmol of an epoxidizing reagent (ethylene glycol diglycidyl ether) were added to 1.0 g of the suction-dried gel, and the mixture was reacted for 16 hours with stirring and shaking in a stirring shaker set to 50°C.
(1-3) After the reaction was completed, the reaction solution was filtered by suction on a glass filter and washed four times with water and four times with 1,4-dioxane to produce a gel into which epoxy groups had been introduced (hereinafter, named Epoxy Toyopearl).

(2) Introduction of haloacetyl groups into Epoxy Toyopearl (2-1) To 1.0 g of Epoxy Toyopearl (suction-dried gel) obtained in (1), 3.5 mmol of an amination reagent (tris(2-aminoethyl)amine) and 1 mL of water were added, and the mixture was reacted for 4 hours with stirring and shaking in a stirring shaker set at 45°C.
(2-2) After the reaction of (2-1) was completed, the reaction solution was filtered by suction on a glass filter and washed four times with water and four times with 1,4-dioxane to prepare a gel into which amino groups had been introduced (hereinafter, named Amino Toyopearl).
(2-3) To 1.0 g of Amino Toyopearl (suction-dried gel), 7.5 mL of 1,4-dioxane, 2.5 mL of water, 0.5 mmol of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), and 0.5 mmol of a haloacetylation reagent (iodoacetic acid) were added, and the mixture was reacted for 2 hours with stirring and shaking in a stirring shaker set at 25°C.
After the reaction of (2-4) and (2-3) was completed, the reaction solution was filtered under suction on a glass filter and washed four times with 1,4-dioxane and four times with water to prepare a gel into which haloacetyl groups had been introduced (hereinafter, referred to as haloacetyl Toyopearl).

Comparative Example 1 Preparation of antibody adsorbent (part 1)
A polypeptide containing the Fc receptor FcR36i, FcR36i_Cys (JP Patent Publication No. 2020-092689) (SEQ ID NO: 4), was immobilized on an insoluble carrier to prepare an antibody adsorbent by the method described below. In FcR36i_Cys (SEQ ID NO: 4), the first methionine to the 22nd alanine is an improved PelB signal peptide (UniProt No. P0C1C1 is an oligopeptide consisting of the 1st to 22nd amino acid residues of P0C1C1, except that the 6th proline is replaced with serine), and the 24th glycine to the 199th glutamine are amino acid residues of the Fc receptor FcR36i (amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, except that the 17th to 192nd amino acid residues are Glu21Gly (this notation indicates that the 21st glutamic acid is replaced with glycine, the same applies below), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51Hi, s, Glu54Asp, Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, Tyr141Phe, Gly147Val, Tyr A polypeptide in which the following amino acid substitutions have been made: Lys158Val, Lys165Glu, Phe171Ser, Val176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp, and Ile190Val, and the sequence from the 201st cysteine to the 207th glycine is a cysteine tag sequence (SEQ ID NO: 3).

(1) Preparation of FcR36i_Cys (1-1) Recombinant Escherichia coli containing the FcR36i_Cys expression vector pTrc-FcR36i_Cys prepared by the method described in JP 2020-092689 A was inoculated into 100 mL of 2xYT liquid medium (tryptone 1.6% (w/v), yeast extract 1% (w/v), sodium chloride 0.5% (w/v)) containing 50 μg/mL kanamycin, and precultured (37°C, 16 hours).
(1-2) 10 mL of the culture solution after preculture was inoculated into 1 L of 2xYT liquid medium containing 50 μg/mL kanamycin and cultured at 37°C for 2 hours. After that, 1 mL of 0.01 M IPTG (isopropyl-β-D-thiogalactopyranoside) was added and further cultured overnight at 25°C with shaking.
(1-3) After completion of the culture, the cells were collected by centrifugation, suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, and disrupted using an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Shoji) at 4° C. for about 10 minutes at an output of about 150 W. The supernatant was then collected by centrifugation.
(1-4) The supernatant collected in (1-3) was added to a column packed with IgG Sepharose 6 Fast Flow (GE Healthcare) equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl (hereinafter referred to as washing buffer), and the column was washed with 10 times the amount of washing buffer compared to the amount of the carrier packed in the column, and 0.1 M glycine-HCl buffer (pH 3.0) was passed through to collect a fraction corresponding to FcR36i_Cys. The eluted fraction was neutralized by adding 1/50 times the amount of 1 M Tris-HCl buffer (pH 9.0) to prepare purified FcR36i_Cys.
(1-5) The absorbance of the collected fractions was measured using a spectrophotometer to quantify the amount of protein contained in the fractions. SDS-PAGE was also performed to confirm that FcR36i_Cys was highly purified.

(2) Preparation of FcR36i_Cys Immobilized Gel (Antibody Adsorbent) (2-1) To 30 mg of FcR36i_Cys (SEQ ID NO: 4) prepared in (1), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to a final concentration of 1 mM, and the mixture was allowed to stand for 1 hour. After that, 1.0 g of Haloacetyl Toyopearl (suction dried gel) prepared in Example 1 and 1 M Tris buffer (pH 9.0) were added, and the mixture was allowed to react at 25° C. for 2 hours.
(2-2) After the reaction was completed, the reaction mixture was washed with 50 mM citrate buffer (pH 3.0), 20 mM MES buffer (pH 6.5), and 20 mM aqueous sodium hydroxide solution, in that order.
(2-3) To 1.0 g of the washed gel (suction-dried gel), 1.8 mL of 20 mM sodium hydroxide, 57 μmol of α-thioglycerol, and 0.18 mL of 1 M Tris buffer (pH 8.5) were added, and the mixture was allowed to react for an additional 2 hours at 25° C. to prepare a gel (antibody adsorbent) on which FcR36i_Cys was immobilized.

Comparative Example 2 Preparation of antibody adsorbent (part 2)
(1) A polypeptide (FcR36i_EAAAK_Cys) (SEQ ID NO: 8) was designed in which a peptide linker consisting of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the 200th glycine and the 201st cysteine of FcR36i_Cys (SEQ ID NO: 4).

(2) PCR was carried out using a polynucleotide (SEQ ID NO: 9) encoding FcR36i_EAAAK_Cys as a template. Oligonucleotides consisting of the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7 were used as primers in the PCR. A reaction solution having the composition shown in Table 1 was prepared using the polynucleotide and primers, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction was then repeated 30 cycles, with one cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute.

(3) The polynucleotide obtained in (2) was purified, digested with the restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrc-PelB (prepared by the method described in WO2015/199154) that had been previously digested with the restriction enzymes NcoI and HindIII, and the ligation product was used to transform the E. coli W3110 strain.

(4) The transformant (genetically modified E. coli) obtained in (3) was cultured (37°C, 16 hours) in 2xYT plate medium (peptone 16 g/L, yeast extract 10 g/L, sodium chloride 5 g/L) containing 50 μg/mL kanamycin, and then the expression vector pTrc-FcR36i_EAAAK_Cys was extracted using a QIAprep Spin Miniprep kit (Qiagen).

(5) Analysis of the nucleotide sequence of pTrc-FcR36i_EAAAK_Cys was performed using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_EAAAK_Cys is shown in SEQ ID NO: 8, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO: 10.

(6) FcR36i_EAAAK_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_EAAAK_Cys obtained in (3) by the method described in Comparative Example 1 (1), and a gel (antibody adsorbent) on which FcR36i_EAAAK_Cys was immobilized was produced by the method described in Comparative Example 1 (2).

Example 2 Preparation of antibody adsorbent (part 3)
(1) A polypeptide (FcR36i_(EAAAK) 3 _Cys) (SEQ ID NO: 11) was designed in which a peptide linker consisting of three repeats of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the 200th glycine and the _201st cysteine of FcR36i_Cys (SEQ ID NO: 4).

(2) PCR was carried out in the same manner as described in Comparative Example 2(2) except that a polynucleotide (SEQ ID NO: 12) encoding FcR36i_(EAAAK) _{3_Cys} was used as the template DNA.

(3) The polynucleotide obtained in (2) was purified, and then ligated and transformed by the method described in Comparative Example 2(3) to obtain recombinant Escherichia coli, and the expression vector pTrc-FcR36i_(EAAAK) ₃ _Cys was extracted by the method described in Comparative Example 2(4).

(4) The nucleotide sequence of pTrc-FcR36i_(EAAAK) ₃ _Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_(EAAAK) ₃ _Cys is shown in SEQ ID NO: 11, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 13.

(5) FcR36i_(EAAAK) ₃ _Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_(EAAAK) 3 _Cys obtained in (3) by the method described in Comparative Example 1 (1), and a gel (antibody adsorbent) _{on which FcR36i_(EAAAK) 3 _Cys} was immobilized was produced by the method described in Comparative Example 1 (2).

Example 3: Preparation of antibody adsorbent (part 4)
(1) A polypeptide (FcR36i_(EAAAK) 5 _Cys) (SEQ ID NO: 14) was designed in which a peptide linker consisting of five repeats of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the 200th glycine and _{the 201st} cysteine of FcR36i_Cys (SEQ ID NO: 4).

(2) PCR was carried out in the same manner as described in Comparative Example 2(2) except that a polynucleotide (SEQ ID NO: 15) encoding FcR36i_(EAAAK) _{5_Cys} was used as the template DNA.

(3) The polynucleotide obtained in (2) was purified, and then ligated and transformed by the method described in Comparative Example 2(3) to obtain recombinant Escherichia coli, and the expression vector pTrc-FcR36i_(EAAAK) ₅ _Cys was extracted by the method described in Comparative Example 2(4).

(4) The nucleotide sequence of pTrc-FcR36i_(EAAAK) ₅ _Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_(EAAAK) ₅ _Cys is shown in SEQ ID NO: 14, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16.

(5) FcR36i_(EAAAK) ₅ _Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_(EAAAK) ₅ _Cys obtained in (3) by the method described in Comparative Example 1 (1), and a gel (antibody adsorbent) on which FcR36i_(EAAAK) ₅ _Cys was immobilized was produced by the method described in Comparative Example 1 (2).

Example 4 Preparation of antibody adsorbent (part 5)
(1) A polypeptide (FcR36i_D1_Cys) was designed in which a peptide linker consisting of the amino acid sequence of SEQ ID NO: 17, which contains the D1 domain of human FcγRIIIa, was inserted between the 200th glycine and the 201st cysteine of FcR36i_Cys (SEQ ID NO: 4) (SEQ ID NO: 18).

(2) PCR was carried out using a polynucleotide (SEQ ID NO: 5) encoding FcR36i_Cys as template DNA. The primers used in the PCR were either one of oligonucleotides (for vector preparation) consisting of sequences set forth in SEQ ID NO: 20 and SEQ ID NO: 21, or oligonucleotides (for insert preparation) consisting of sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23. A reaction solution having the composition shown in Table 1 was prepared using the template DNA and primers, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction was carried out by repeating 30 cycles of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 5 minutes.

(3) The two types of polynucleotides obtained in (2) were each purified, and a mixed solution containing 0.03 pmol of vector polynucleotide and 0.06 pmol of insert polynucleotide was prepared. The mixed solution was ligated to the vector polynucleotide and the insert polynucleotide by Gibson assembly reaction using NEB Builder HiFi DNA Assembly Master Mix (New England Biolabs), and the ligation product was used to transform E. coli W3110 strain.

(4) The expression vector pTrc-FcR36i_D1_Cys was extracted from the transformant (genetically modified E. coli) obtained in (3) by the method described in Comparative Example 2 (4).

(5) The nucleotide sequence of pTrc-FcR36i_D1_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_D1_Cys is shown in SEQ ID NO: 18, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO: 19.

(6) FcR36i_D1_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_D1_Cys obtained in (3) by the method described in Comparative Example 1 (1), and a gel (antibody adsorbent) on which FcR36i_D1_Cys was immobilized was produced by the method described in Comparative Example 1 (2).

Example 5 Preparation of antibody adsorbent (part 6)
(1) A polypeptide (FcR36i_2D1_Cys) (SEQ ID NO: 24) was designed in which a peptide linker consisting of two repeats of the amino acid sequence of SEQ ID NO: 17 was inserted between the 200th glycine and the 201st cysteine of FcR36i_Cys (SEQ ID NO: 4).

(2) PCR was performed in the same manner as described in Example 4(2), except that a polynucleotide encoding FcR36i_D1_Cys (SEQ ID NO: 19) was used as the template DNA, and either one of the oligonucleotides having the sequences described in SEQ ID NO: 20 and SEQ ID NO: 26 (for vector preparation) or the oligonucleotides having the sequences described in SEQ ID NO: 22 and SEQ ID NO: 27 (for insert preparation) was used as the PCR primers.

(3) After purifying each of the two types of polynucleotides obtained in (2), they were ligated and transformed by the method described in Example 4 (3) to obtain recombinant E. coli, and the expression vector pTrc-FcR36i_2D1_Cys was extracted by the method described in Comparative Example 2 (4).

(4) The nucleotide sequence of pTrc-FcR36i_2D1_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_2D1_Cys is shown in SEQ ID NO: 24, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO: 25.

(5) FcR36i_2D1_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_2D1_Cys obtained in (3) by the method described in Comparative Example 1 (1), and a gel (antibody adsorbent) on which FcR36i_2D1_Cys was immobilized was produced by the method described in Comparative Example 1 (2).

Example 6 Measurement of antibody adsorption amount (part 1)
(1) The antibody adsorbents prepared in Comparative Examples 1 and 2 and Examples 2 and 3 were suction-dried by the method described in Example 1 (1-1) to prepare suction-dried gels.

(2) 150 μL of PBS (Phosphate Buffered Saline) was added to the suction-dried gel and centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to wash the gel.

(3) After washing, 150 μL of PBS and 60 μL of human immunoglobulin solution (globulin intramuscular injection 1500 mg/10 mL "JB", manufactured by Japan Blood Products Organization) were added sequentially to the gel, and the gel was stirred at 25°C for 2 hours to allow the immunoglobulin (antibody) to be adsorbed onto the gel.

(4) After the adsorption procedure in (3), the spin column was centrifuged at 2000 × g for 1 minute to separate the solution containing unadsorbed antibody from the gel.

(5) 150 μL of PBS was added to the gel and centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to wash the gel.

(6) 150 μL of 50 mM citrate buffer (pH 3.0) was added to the gel, and the gel was centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to elute the antibodies adsorbed to the gel. The absorbance of the eluate was measured to calculate the antibody concentration, and the amount of antibody adsorbed to the Fc receptor-immobilized gel was determined.

The results are shown in Table 2. The linker lengths in Table 2 are shown for reference based on values given in the literature (Protein Engineering, 14(8), 529-532 (2001)). It can be seen that the antibody adsorption amount relative to the amount of FcR immobilized in the antibody adsorbents with linker lengths of 2 nm or more (Examples 2 and 3) is improved compared to the antibody adsorbents without a linker (Comparative Example 1) and those with linker lengths of less than 2 nm (Comparative Example 2).

Example 7 Measurement of antibody adsorption amount (part 2)
The antibody adsorption amount was measured in the same manner as described in Example 6, except that the antibody adsorbents prepared in Comparative Example 1 and Examples 4 and 5 were used instead of the antibody adsorbents in Example 6(1).

The results are shown in Table 3. The linker length in Table 3 indicates the approximate value of the D1 domain length of FcγRIIIa calculated from the crystal structure (Protein databank: 3AY4) containing the Fc receptor. Even if the peptide linker sequence is not a simple repetition of amino acid sequences as in Examples 2 and 3, but is a domain sequence of a specific protein, it can be seen that the amount of antibody adsorption relative to the amount of FcR immobilized is improved as long as the linker length is 2 nm or more, compared to the antibody adsorbent without a linker (Comparative Example 1).

Claims (2)

An antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier,
The Fc receptor is a polypeptide comprising 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:4,
The peptide linker is a polypeptide represented by any one of the following (1) or (2):
(1) A polypeptide comprising at least the amino acid residues from the 6th proline to the 87th histidine in the amino acid sequence set forth in SEQ ID NO: 17;
(2) A polypeptide consisting of a sequence in which the amino acid sequence set forth in SEQ ID NO:2 is repeated three or more times.
and
The adsorbent, wherein the length of the peptide linker is 2 nm or more. 13. A method for purifying an antibody, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent according to claim 1 to adsorb the antibody onto the adsorbent; and eluting the antibody adsorbed onto the adsorbent using an elution solution.