JP7524591B2 - Insoluble carrier capable of immobilizing proteins and method for producing same - Google Patents

Description

The present invention relates to an insoluble carrier capable of immobilizing proteins and a method for producing the same. More specifically, the present invention relates to an insoluble carrier capable of immobilizing a larger amount of proteins by optimizing the amount of activated functional groups possessed by the insoluble carrier, and a method for producing the same.

Antibody drugs are medicines that use antibodies (immunoglobulins), molecules that are responsible for the immune function in the body. Antibody drugs bind to target molecules with high specificity and affinity due to the diversity of the variable regions of antibodies. 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.

The manufacture of antibody drugs involves a culture process and a purification process. In the culture process, efforts are made to improve productivity by modifying the antibody-producing cells and optimizing the culture conditions. In the purification process, affinity chromatography is used for crude purification, and the drug is then formulated after intermediate purification, final purification, and virus removal.

In the above-mentioned affinity chromatography, an affinity carrier that specifically recognizes antibody molecules is used. To mass-produce antibody drugs at low cost, it is necessary to improve the amount of antibody drugs adsorbed to the affinity carrier. To achieve this, it is necessary to produce an affinity carrier on which an appropriate amount of ligand capable of specifically binding to the antibody drug is immobilized.

In order to immobilize an appropriate amount of a ligand capable of specifically binding to an antibody drug on an insoluble carrier, it is necessary to optimize the activated functional groups capable of immobilizing the ligand on the insoluble carrier. Patent Document 1 discloses an insoluble carrier having 300 μmol to 3000 μmol of activated functional groups per gram of the carrier in a dry state as an insoluble carrier for immobilizing a substance that exhibits biological affinity. However, depending on the substance that exhibits biological affinity to be immobilized, the above conditions may not necessarily be the optimal insoluble carrier for immobilizing the substance.

Japanese Unexamined Patent Publication No. 178106/1983

The objective of the present invention is to provide an insoluble carrier capable of immobilizing an antibody-binding protein in order to produce an antibody adsorbent that can also be used for the purpose of separating antibodies.

In order to solve the above problems, the inventors conducted extensive research and found that by optimizing the amount of activated functional groups used to immobilize antibody-binding proteins on insoluble carriers, they were able to improve the amount of antibody adsorption compared to conventional methods.

That is, the present invention includes the following aspects [1] to [6].

[1] An insoluble carrier having an activated functional group capable of immobilizing a protein, the protein being an antibody-binding protein, and the amount of the activated functional group possessed by the insoluble carrier being 2.5 μmol or more and 40 μmol or less per 1 g of the insoluble carrier.

[2] The insoluble carrier according to [1], wherein the antibody-binding protein is a human Fcγ receptor.

[3] A human Fcγ receptor,
(1) 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, or (2) 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, further comprising 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, and having IgG binding activity.
The insoluble carrier according to [2].

[4] An insoluble carrier according to any one of [1] to [3], wherein the activated functional group is a haloacetyl group or a maleimide group.

[5] A method for producing an insoluble carrier according to any one of [1] to [4], comprising the steps of introducing an epoxy group into an insoluble carrier having a hydroxy group, and introducing an activated functional group capable of immobilizing a protein to the epoxy group.

[6] A method for producing an antibody adsorbent, comprising a step of immobilizing an antibody-binding protein to an activated functional group possessed by an insoluble carrier described in any one of [1] to [4].

The present invention is described in detail below.

Examples of antibody-binding proteins in the present invention include proteins that can specifically bind to the Fc region, Fab region, and glycans of antibodies (immunoglobulins). Among these, Fc-binding proteins, which are proteins that can specifically bind to the Fc region, are preferred.

Examples of Fc-binding proteins include Protein A and Protein G derived from the genus Staphylococcus, Fc receptors, and partial regions of the Fc receptors having Fc-binding ability. Among these, when the insoluble carrier of the present invention is intended to produce an IgG adsorbent containing a human Fc region, human Fcγ receptors are preferred. Specific examples of human Fcγ receptors include human FcγRI, human FcγRIIa, human FcγRIIb, human FcγRIIIa, and human FcRn. In particular, human FcγRIIIa is a human Fcγ receptor that can recognize the sugar chain structure of an antibody, and an antibody adsorbent 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), making it a preferred embodiment of the human Fcγ receptor to be immobilized on the insoluble carrier of the present invention.

A preferred embodiment of the human FcγRIIIa is
(1) A polypeptide comprising at least the extracellular region of native human FcγRIIIa (amino acid residues from glycine at position 17 to methionine at position 192 of the amino acid sequence set forth in SEQ ID NO:1), and (2) a polypeptide comprising at least the extracellular region of native human FcγRIIIa, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions among the amino acid residues constituting the extracellular region, and having antibody binding activity.
In addition, a more preferred embodiment of the above (2) 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:

The insoluble carrier of the present invention may be any material that has an activated functional group capable of immobilizing an antibody-binding protein, as described below, and is insoluble in a solution or solvent used for adsorption/elution of the antibody. The insoluble carrier may be a carrier derived from an inorganic material such as zirconia, zeolite, silica, or coated silica, a carrier derived from a natural organic polymeric material such as cellulose, agarose, or dextran, or a carrier derived from a synthetic organic polymeric 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), Sepharose (GE Healthcare), Amberlite (DuPont), Cellufine (JNC), POROS (Thermo Fisher Scientific), or Toyopearl (Tosoh).

Examples of activated functional groups capable of immobilizing antibody-binding proteins include activated functional groups such as N-hydroxysuccinimide (NHS) activated ester groups, carboxy groups, maleimide groups, haloacetyl groups, tresyl groups, formyl groups, and haloacetamide groups. Among these, haloacetyl groups and maleimide groups are preferred.

Methods for introducing an activating functional group onto the surface of an insoluble carrier include a method of introducing a compound having an activating functional group to a hydroxy group, epoxy group, carboxy group, amino group, or the like on the surface of the carrier. Among these, a method of introducing an epoxy group into an insoluble carrier having a hydroxy group and then introducing the activating functional group onto the epoxy group is preferred.

Examples of compounds (hereinafter referred to as epoxidizing reagents) that introduce epoxy groups to hydroxy groups on the surface of an insoluble carrier include epihalohydrins such as epichlorohydrin and epibromohydrin, diglycidyl ethers such as ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, polyethylene glycol diglycidyl ether, and bisphenol A diglycidyl ether, and alkyl diene diepoxides such as 1,7-octadiene diepoxide. Of these, it is preferable to use polyethylene glycol diglycidyl ether having a repeating unit of -C ₂ H ₄ O- of 1 to 10. Epoxy groups can be introduced by reacting them in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate, or in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate containing sodium borohydride.

If a carboxyl group is to be introduced as an activated functional group after the epoxy group is introduced onto the carrier surface using the aforementioned epoxidizing reagent, then the introduction can be performed using a carboxylizing reagent such as 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, or 4-aminobutyric acid. In addition, when introducing an amino group as an activated functional group, for example, an amination reagent such as ethylenediamine, 1,3-propanediamine, putrescine, cadaverine, hexamethylenediamine, 1,8-octanediamine, 1,10-decanediamine, 1,12-dodecanediamine, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, trans-1,4-cyclohexanediamine, trans-1,2-cyclohexanediamine, 4,4'-diaminodiphenyl ether, 4,4'-ethylenedianiline, spermidine, spermine, bis(hexamethylene)triamine, N,N'-bis(2-aminoethyl)-1,3-propanediamine, N,N'-bis(3-aminopropyl)ethylenediamine, triethylene glycol bis(2-aminoethyl)ether, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, or tris(4-aminophenyl)amine may be used for the introduction.

When introducing a maleimide group as an activated functional group to an epoxy group, hydroxy group, carboxy group, or amino group on the surface of an insoluble carrier, for example, 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, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, This can be introduced using a maleimidation reagent such as carboxylic 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, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, etc.

When introducing a haloacetyl group as an activated functional group into an epoxy group, hydroxy group, or amino group on the surface of an insoluble carrier, for example, a haloacetylation reagent such as 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, or 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester may be used. Another embodiment of the method for introducing a haloacetyl group is to react an ω-alkenylalkane glycidyl ether with an epoxy group, hydroxy group, or amino group on the surface of an insoluble carrier, and then halogenate the ω-alkenyl moiety 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 the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

When the insoluble carrier surface has a carboxyl group, there is also a method of introducing an activated functional group into the carboxyl group using a condensation agent and an additive. Examples of the condensation agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

Water is usually used as a reaction solvent when introducing functional groups onto the surface of an insoluble carrier, but other solvents may also be used, such as hydrocarbons such as n-hexane, benzene, and xylene, ethers such as tetrahydrofuran and dioxane, halogenated hydrocarbons such as chloroform and chlorobenzene, alcohols such as ethanol and methyl cellosolve, ketones such as acetone and methyl isobutyl ketone, and 1,4-dioxane, dimethylformamide, and dimethyl sulfoxide. These solvents may be used alone or in a mixed solvent system. When introducing functional groups onto the surface of an insoluble carrier, no particular catalyst is required, but hydroxides or carbonates of alkali or alkaline earth metals such as sodium hydroxide and potassium carbonate may be used as catalysts.

The insoluble carrier of the present invention is characterized in that the amount of activated functional groups capable of immobilizing antibody-binding proteins is 2.5 μmol to 40 μmol per gram of insoluble carrier, and preferably 3 μmol to 30 μmol. In this specification, the weight of the insoluble carrier is the weight as a suction dry gel.

An antibody adsorbent can be produced by immobilizing an antibody-binding protein on the insoluble carrier of the present invention. One example of such a production method is a method in which the antibody-binding protein is immobilized by covalent bonding via an activated functional group possessed by the insoluble carrier. Examples of buffer solutions used when immobilizing an antibody-binding protein 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 a temperature range of 5°C to 50°C, taking into consideration the reactivity of the functional group and the stability of the ligand, and is preferably in the range of 10°C to 35°C.

The present invention relates to optimizing the amount of activated functional groups possessed by an insoluble carrier. The insoluble carrier of the present invention makes it possible to produce an antibody adsorbent with an improved antibody adsorption amount compared to conventional methods. Therefore, the present invention can contribute to the production of antibody separation columns used in the industrial production of antibody pharmaceuticals.

Each embodiment of the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples in any way.

Example 1 Preparation of antibody-binding protein immobilized gel (1) Introduction of epoxy groups into gel having hydroxyl groups (1-1) A slurry of polymethacrylate gel (Toyopearl, manufactured by Tosoh Corporation) having hydroxyl groups on its surface was suction filtered on a glass filter and then dried by suction to prepare a suction-dried gel.
(1-2) 5 mL of 1,4-dioxane, 0.5 mL of a 0.2 M aqueous sodium hydroxide solution, and an epoxidizing reagent having a final concentration of 0.45 mmol to 18 mmol were added to 1.0 g of the suction-dried gel, and the mixture was reacted with stirring and shaking in a stirring shaker set to 50° C. for 16 hours or more.
(1-3) After the reaction was completed, the reaction solution was filtered through a glass filter under suction 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), 2 mL of 1,4-dioxane or 1 mL of water was added, and the mixture was reacted with stirring and shaking for 4 to 24 hours in a stirring shaker set at 45°C.
(2-2) After the reaction of (2-1) was completed, the reaction solution was suction filtered 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, EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) at a final concentration of 0.5 mmol, and a haloacetylation reagent (iodoacetic acid) at a final concentration of 0.5 mmol were added, and the mixture was reacted with stirring and shaking for 2 hours 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).

(3) Introduction of Antibody-Binding Protein (3-1) Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to 30 mg of the antibody-binding protein to a final concentration of 1 mM, and the mixture was allowed to stand for 1 hour. After that, 1.0 g of the haloacetyl Toyopearl (suction-dried gel) prepared in (2) and 1 M Tris buffer (pH 7.0) were added and reacted for 2 hours at 25° C. The Fc-binding protein FcR36i_Cys (SEQ ID NO: 2) disclosed in WO2019/083048 was used as the antibody-binding protein. In FcR36i_Cys (SEQ ID NO: 2), the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 24th glycine (Gly) to the 199th glutamine (Gln) is the amino acid sequence of the Fc binding protein FcR36i, and the portion from the 200th glycine (Gly) to the 207th glycine (Gly) is the cysteine tag sequence. The FcR36i is 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), with the following amino acid residues in the region: Glu21Gly (this notation indicates that glutamic acid at position 21 of SEQ ID NO: 1 (corresponding to position 28 of SEQ ID NO: 2) is replaced with glycine, the same applies below), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51His, Glu54Asp, Asn56Asp, The polypeptide has the amino acid substitutions Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, Tyr141Phe, Gly147Val, Tyr158Val, Lys165Glu, Phe171Ser, Val/Phe176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp and Ile190Val.
(3-2) 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.
(3-3) 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 in which the antibody-binding protein was immobilized (hereinafter referred to as a ligand-immobilized gel).

Example 2 Measurement of the Amount of Haloacetyl Groups (1) Phosphate buffered saline (PBS) (pH 7.4) was added to 1 mL of the ligand-immobilized gel prepared in Example 1 to give a 50% slurry.

(2) After homogenizing the prepared slurry, 100 μL of the slurry (50 μL as immobilized gel) was added to a spin column (Cosmos Spin Filter H (pore size 0.45 μm), manufactured by Nacalai Tesque) and centrifuged at 2000 rpm for 1 minute to prepare a suction-dried gel.

(3) 50 μL of 20 mM α-thioglycerol, 30 μL of PBS, and 20 μL of 1 M Tris buffer (pH 9.0) were added to the suction-dried gel and stirred at 40°C for 1 hour.

(4) The reaction solution was collected as a filtrate by centrifuging at 2000 rpm for 1 minute.

(5) 940 μL of phosphate buffer (pH 8.0) and 20 μL of 20 mM 2,2'-dipyridyl disulfide were added to 50 μL of the filtrate recovered in (4), and the absorbance was measured. The amount of unreacted α-thioglycerol was calculated from the absorbance of 2-thiopyrrolidone produced by the reductive decomposition of excess α-thioglycerol by 2,2'-dipyridyl disulfide, and the amount of α-thioglycerol that reacted with the haloacetyl groups was calculated back to quantify the haloacetyl groups.

Example 3 Measurement of antibody adsorption amount (1) A suction dry gel was prepared in the same manner as in Example 2 (1) and (2).

(2) 150 μL of PBS was added to the suction-dried gel and centrifuged at 2000 rpm 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.

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

(6) 150 μL of PBS was added to the gel and centrifuged at 2000 rpm for 1 minute. This procedure was repeated three times to wash the gel.

(7) 150 μL of 50 mM citrate buffer (pH 3.0) was added to the gel, and the gel was centrifuged at 2000 rpm for 1 minute. This procedure was repeated three times to elute the antibodies adsorbed to the gel. The antibody concentration was calculated by measuring the absorbance of the eluate, and the amount of antibody adsorbed to the ligand-immobilized gel was determined.

The results are shown in Tables 1 and 2. In Table 1, "epoxidation reagent" refers to the epoxidation reagent used in Example 1 (1-2). In the "Evaluation" column, the antibody adsorption amount per 1 g of ligand-immobilized gel is marked as "○" if it is 35 mg or more, "△" if it is 30 mg to 35 mg, and "×" if it is 30 mg or less. All of the ligand-immobilized gels (ligand-immobilized gels 1 to 16) with an amount of activated functional groups of 2.5 μmol to 40 μmol per 1 g of insoluble carrier showed good antibody adsorption amounts. On the other hand, the ligand-immobilized gels (ligand-immobilized gels 17 to 19) with an amount of activated functional groups of less than 2.5 μmol per 1 g of insoluble carrier did not have a sufficient amount of activated functional groups and had significantly fewer antibody adsorption sites, so sufficient antibody adsorption amounts were not obtained.

Claims (4)

An insoluble carrier having an activated functional group for immobilizing a protein,
the protein is a human Fcγ receptor ;
the activating functional group is a haloacetyl group;
the insoluble carrier is a polymethacrylate gel;
The insoluble carrier has an amount of activated functional groups of 2.5 μmol or more and 40 μmol or less per 1 g of the insoluble carrier. Human Fcγ receptor,
(1) 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, or (2) 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, further comprising 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, and having IgG binding activity.
The insoluble carrier according to claim 1 . A step of introducing an epoxy group into an insoluble carrier having a hydroxy group;
and introducing an activated functional group capable of immobilizing a protein onto the epoxy group. A method for producing an antibody adsorbent, comprising the step of immobilizing a human Fcγ receptor to an activated functional group possessed by the insoluble carrier according to claim 1 or 2 .