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WO2025173342A1 - Support chromatographique pour purifier une protéine de fusion fc et procédé de purification de protéine de fusion fc l'utilisant - Google Patents

Support chromatographique pour purifier une protéine de fusion fc et procédé de purification de protéine de fusion fc l'utilisant

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Publication number
WO2025173342A1
WO2025173342A1 PCT/JP2024/041646 JP2024041646W WO2025173342A1 WO 2025173342 A1 WO2025173342 A1 WO 2025173342A1 JP 2024041646 W JP2024041646 W JP 2024041646W WO 2025173342 A1 WO2025173342 A1 WO 2025173342A1
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WO
WIPO (PCT)
Prior art keywords
porous particles
fusion protein
ligand
protein
meth
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Pending
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PCT/JP2024/041646
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English (en)
Japanese (ja)
Inventor
喬太 鈴木
政完 柳
イン ヤオ
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JSR Corp
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JSR Corp
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Publication of WO2025173342A1 publication Critical patent/WO2025173342A1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to a chromatography support for purifying Fc fusion proteins and a method for purifying Fc fusion proteins using the support.
  • the target pharmaceutical protein such as an antibody
  • the target pharmaceutical protein must be separated from the expression medium and purified to a purity acceptable for use as a therapeutic or diagnostic agent.
  • Pharmaceutical proteins are generally produced through purification using affinity chromatography.
  • the affinity support used in this type of chromatographic purification is required to have an improved binding capacity for the target protein and reduced non-specific adsorption of impurities such as culture medium components.
  • Fc fusion proteins are proteins in which the Fc fragment of immunoglobulin is fused with a protein (such as the extracellular domain of a receptor) that has the ability to specifically bind to a target molecule, and are used clinically as molecularly targeted therapeutics.
  • Fc fusion proteins are purified by affinity chromatography, similar to antibody drugs.
  • affinity chromatography similar to antibody drugs.
  • conventional affinity supports are manufactured primarily for the purpose of purifying antibodies, their use in purifying Fc fusion proteins did not achieve performance equivalent to that of antibody purification in terms of dynamic binding capacity (DBC) or impurity contamination. It is desirable to separate Fc fusion proteins with higher efficiency and purity.
  • DBC dynamic binding capacity
  • the present invention provides a chromatography support that enables the separation of Fc fusion proteins with a large DBC and fewer impurities, and a method for purifying Fc fusion proteins using the same.
  • a chromatography support for purifying an Fc fusion protein comprising: Porous particles having ligands immobilized thereon, the porous particles are synthetic polymer-based or natural polymer-based porous particles, the ligand is at least one selected from the group consisting of protein A, protein G, protein L, and analogs thereof; the porous particles to which the ligands are immobilized have an average pore size of 75 to 120 nm; Chromatography support.
  • the chromatography carrier according to [1], wherein the porous particles to which the ligands are immobilized have an average pore size of 101 to 110 nm.
  • the porous particles are a polymer of a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group, and a polymerizable unsaturated group-containing monomer that does not have the functional group.
  • an "Fc fusion protein” refers to a protein in which a heterologous protein is fused to the Fc fragment of an immunoglobulin.
  • the heterologous protein may be of human or non-human origin, but is a protein other than immunoglobulin Fab.
  • Fc fusion proteins are distinguished from immunoglobulins such as IgG, IgA, IgD, IgE, and IgM, as well as antibodies and fragments thereof, including chimeric antibodies.
  • the heterologous protein is a protein capable of specifically binding to a target molecule such as the extracellular domain of a receptor, a receptor antagonist, or an enzyme.
  • Fc fusion proteins are used as molecularly targeted therapeutic agents for the treatment of inflammatory diseases such as rheumatoid arthritis and bleeding disorders such as hemophilia.
  • inflammatory diseases such as rheumatoid arthritis and bleeding disorders such as hemophilia.
  • molecularly targeted therapeutic agents using Fc fusion proteins include etanercept, rilonacept, and efmoroctocog alfa.
  • amino acid sequence identity preferably refers to 90% or greater identity, more preferably 94% or greater, even more preferably 96% or greater, even more preferably 98% or greater, and even more preferably 99% or greater identity.
  • Amino acid sequence identity can be determined using the BLAST algorithm (Pro. Natl. Acad. Sci. USA., 1993, 90:5873-5877). Based on this BLAST algorithm, programs called BLASTN, BLASTX, BLASTP, TBLASTN, and TBLASTX have been developed (J. Mol. Biol., 1990, 215:403-410). When using these programs, the default parameters of each program can be used. Specific techniques for these analysis methods are known (see [www.ncbi.nlm.nih.gov]).
  • a "corresponding position" in an amino acid sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology between conserved amino acid residues present in each amino acid sequence. Alignment can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson J.D. et al., Nucleic Acids Res., 1994, 22:4673-4680) with default settings.
  • Clustal W is available, for example, on the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp/index.html]), operated by the National Institute of Genetics.
  • DDBJ DNA Data Bank of Japan
  • a position in a target sequence that is aligned to a given position in a reference sequence by the above-described alignment is considered to be a "position corresponding to" that given position.
  • amino acid sequence of a peptide is written in accordance with conventional practice, with the amino terminus (hereinafter referred to as the N-terminus) on the left and the carboxyl terminus (hereinafter referred to as the C-terminus) on the right.
  • the cyclic ether group may have an alkyl group as a substituent.
  • Examples of cyclic ether groups include cyclic ether groups represented by the following formulas (1) to (6). Of these, cyclic ether groups represented by formulas (1), (3), or (6) are preferred, with the cyclic ether group represented by formula (1) being more preferred.
  • the total content of the functional group-containing monomers in the monomer composition is preferably 35 parts by mass or more, more preferably 45 parts by mass or more, and even more preferably 55 parts by mass or more, per 100 parts by mass of the total amount of monomers in the monomer composition; and is preferably 99 parts by mass or less, more preferably 90 parts by mass or less, and even more preferably 85 parts by mass or less, per 100 parts by mass of the total amount of monomers in the monomer composition.
  • the monomer composition may further contain a monomer other than the functional group-containing monomer (hereinafter also referred to as "other monomer").
  • a monomer other than the functional group-containing monomer hereinafter also referred to as "other monomer”
  • examples of such other monomers include polymerizable unsaturated group-containing monomers that do not have a functional group capable of immobilizing a ligand.
  • Such other monomers are broadly classified as non-crosslinkable monomers and crosslinkable monomers, and either one of these may be used alone or in combination.
  • non-crosslinkable monomers examples include (meth)acrylate-based non-crosslinkable monomers, (meth)acrylamide-based non-crosslinkable monomers, aromatic vinyl-based non-crosslinkable monomers, vinyl ketone-based non-crosslinkable monomers, (meth)acrylonitrile-based non-crosslinkable monomers, and N-vinylamide-based non-crosslinkable monomers. These can be used alone or in combination of two or more. Of these non-crosslinkable monomers, (meth)acrylate-based non-crosslinkable monomers and aromatic vinyl-based non-crosslinkable monomers are preferred.
  • Examples of the (meth)acrylate-based non-crosslinkable monomers include methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono(meth)acrylate, trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, butanetriol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, pentaerythritol mono
  • N-vinylamide non-crosslinkable monomer examples include N-vinylacetamide and N-vinylpropionamide. These can be used alone or in combination of two or more.
  • the total content of the non-crosslinkable monomers in the monomer composition is preferably 0.01 parts by mass or more, more preferably 0.05 parts by mass or more, and even more preferably 0.1 parts by mass or more, per 100 parts by mass of the total amount of monomers in the monomer composition; and is preferably 30 parts by mass or less, more preferably 15 parts by mass or less, and even more preferably 5 parts by mass or less, per 100 parts by mass of the total amount of monomers in the monomer composition.
  • Crosslinking monomers that can be used as the other monomers include, for example, (meth)acrylate-based crosslinking monomers, aromatic vinyl-based crosslinking monomers, and allyl-based crosslinking monomers. These can be used alone or in combination of two or more. Di- to penta-functional crosslinking monomers are preferred as the crosslinking monomer, with di- or tri-functional crosslinking monomers being more preferred. Among these crosslinking monomers, (meth)acrylate-based crosslinking monomers and aromatic vinyl-based crosslinking monomers are preferred.
  • acrylates include butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(
  • allyl crosslinkable monomer examples include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, triallyl isocyanurate, etc. These may be used alone or in combination of two or more.
  • examples of the crosslinkable monomer include dehydration condensation reaction products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.
  • the total content of the crosslinkable monomers in the monomer composition is preferably 1 part by mass or more, more preferably 5 parts by mass or more, and even more preferably 10 parts by mass or more, per 100 parts by mass of the total amount of monomers in the monomer composition; and is preferably 50 parts by mass or less, more preferably 40 parts by mass or less, and even more preferably 30 parts by mass or less, per 100 parts by mass of the total amount of monomers in the monomer composition.
  • the porosifying agent may, for example, be an aliphatic hydrocarbon such as hexane, heptane, octane, nonane, decane, or undecane; an alicyclic hydrocarbon such as cyclopentane or cyclohexane; an aromatic hydrocarbon such as benzene, toluene, xylene, naphthalene, ethylbenzene, or polystyrene; a halogenated hydrocarbon such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, or chlorobenzene; an aliphatic alcohol such as butanol, pentanol, hexanol, heptanol, 4-methyl-2-pentanol, or 2-ethyl-1-hexanol; an alicyclic alcohol such as cyclohexanol; 2-phenylethyl alcohol, benz
  • Specific methods for producing the porous particles used in the present invention include, for example, a method in which a polymerization initiator is dissolved in a mixed solution (monomer solution) containing the monomer composition and porosifying agent, the mixture is suspended in the aqueous medium, and the mixture is heated to a predetermined temperature to polymerize; a method in which a polymerization initiator is dissolved in a mixed solution (monomer solution) containing the monomer composition and porosifying agent, the mixture is added to the aqueous medium heated to a predetermined temperature to polymerize; and a method in which a mixed solution (monomer solution) containing the monomer composition and porosifying agent is suspended in the aqueous medium, heated to a predetermined temperature, and a polymerization initiator is added to polymerize.
  • a variety of surfactants may be used to produce the porous particles, including anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfate ester salts, alkyl phosphate ester salts, and fatty acid salts.
  • Polymerization inhibitors may also be used, including nitrites such as sodium nitrite, iodide salts such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride.
  • Polymerization regulators such as dodecyl mercaptan may also be used.
  • the polymerization temperature of the monomer can be determined depending on the polymerization initiator, but is typically around 2 to 100°C, with 50 to 100°C being preferred.
  • the polymerization time of the monomer is typically 5 minutes to 48 hours, and preferably 10 minutes to 24 hours.
  • the resulting porous particles may be reacted with at least one agent selected from the group consisting of a crosslinking agent and a hydrophilizing agent.
  • a crosslinking agent and a hydrophilizing agent are used, the crosslinking reaction may be performed after the hydrophilizing reaction, or after the hydrophilizing reaction.
  • the crosslinking reaction and the hydrophilizing reaction may be performed simultaneously.
  • the crosslinking reaction and the hydrophilizing reaction of the porous particles may each be performed in parallel with the polymerization reaction of the monomers using the polymerization initiator.
  • the crosslinking reaction involves an addition reaction of the crosslinking agent with some of the functional groups present in the polymer molecules of the porous particles, introducing a partial structure derived from the crosslinking agent. This crosslinks the residues of the functional groups via the partial structure derived from the crosslinking agent.
  • the hydrophilizing reaction involves an addition reaction of the hydrophilizing agent with some of the functional groups present in the polymer molecules of the porous particles, introducing a partial structure derived from the hydrophilizing agent.
  • the crosslinking agent used in the crosslinking reaction may be any agent capable of reacting with a functional group capable of immobilizing a ligand to introduce a crosslinked structure.
  • dicarboxylic acid dihydrazides and (alkylenebisimino)bis(oxoalkanoic acids) are preferred, with dicarboxylic acid dihydrazides being more preferred, in order to improve the liquid permeability of the chromatography support, or the pressure resistance characteristics and antifouling properties during liquid passage.
  • crosslinking agents other than those mentioned above can also be used.
  • crosslinking agents include polyfunctional isocyanate-based crosslinking agents, polyfunctional epoxy-based crosslinking agents, polyfunctional aldehyde-based crosslinking agents, polyfunctional thiol-based crosslinking agents, polyfunctional oxazoline-based crosslinking agents, polyfunctional aziridine-based crosslinking agents, and metal chelate-based crosslinking agents.
  • the total amount of the crosslinking agent used in the crosslinking reaction is preferably 0.01 molar equivalents or more, more preferably 0.05 molar equivalents or more, and even more preferably 0.1 molar equivalents or more, relative to 1 mole of functional group derived from the functional group-containing monomer used in producing the porous particles, and is preferably 0.8 molar equivalents or less, more preferably 0.7 molar equivalents or less, and even more preferably 0.6 molar equivalents or less.
  • hydrophilizing agent used in the hydrophilization reaction in order to improve the antifouling properties or low ligand leakage properties of the chromatography support, a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule is preferred, and a compound having a total of 2 to 4 hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule is more preferred.
  • hydrophilizing agents include alcohols having a mercapto group in the molecule, such as mercaptoethanol and thioglycerol; and polyhydric alcohols such as glycerol and diglycerol.
  • hydrophilizing agents can be used alone or in combination of two or more. Of these, in order to improve the antifouling properties or low ligand leakage properties of the support, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is more preferred.
  • the total amount of the hydrophilizing agent used in the hydrophilization reaction is preferably 0.5 molar equivalents or more, more preferably 1 molar equivalent or more, and even more preferably 2 molar equivalents or more, relative to 1 mole of functional group derived from the functional group-containing monomer used in producing the porous particles, and is preferably 10 molar equivalents or less, more preferably 8 molar equivalents or less, and even more preferably 6 molar equivalents or less.
  • Protein A contains five domains, E, D, A, B, and C, which have the ability to bind to the Fc of immunoglobulin and are therefore Fc-binding domains.
  • the protein A analog used as a ligand in the present invention is an Fc-binding protein containing the protein A Fc-binding domain or a modified version thereof.
  • the ligand of the present invention contains one or more Fc-binding domains selected from the group consisting of the protein A B domain, the protein A C domain, or modified versions thereof. More preferably, the ligand contains the protein A C domain consisting of the amino acid sequence of SEQ ID NO: 1, or one or more Fc-binding domains selected from the group consisting of modified versions thereof. Even more preferably, the Fc-binding domain contained in the ligand is a modified version of the protein A C domain consisting of the amino acid sequence of SEQ ID NO: 1.
  • the Fc-binding domain contained in the ligand consists of an amino acid sequence in which three or more substitutions, more preferably three to nine substitutions, even more preferably three to six substitutions, and even more preferably three to five substitutions selected from the group consisting of (a) to (i) above have been made relative to the amino acid sequence of the parent Fc-binding domain.
  • substitutions include: A combination of (a), (b) and (g); A combination of (a), (c), and (g); A combination of (a), (b), (c), and (g); A combination of (a), (d), and (g); A combination of (a), (e), and (g); A combination of (d), (e), and (h); A combination of (a), (e), (f), and (g); A combination of (a), (e), (g), and (i); A combination of (a), (b), (c), (e), and (g); A combination of (a), (b), (c), (e), (g), and (i).
  • the host for transformation is not particularly limited, and known hosts used to express recombinant proteins, such as bacteria such as Escherichia coli, fungal cells, insect cells, and mammalian cells, can be used.
  • bacteria such as Escherichia coli, fungal cells, insect cells, and mammalian cells
  • any method known in the art can be used depending on the host. For example, known methods such as those described in Molecular Cloning (edited by Sambrook et al.) (Cold Spring Harbor Laboratory Press, 3rd edition, 2001) can be used.
  • the ligand can be immobilized on the porous particles via a linker (spacer).
  • a linker spacer
  • Introduction of a linker to a porous particle and immobilization of a ligand using a linker can be performed with reference to U.S. Patent No. 5,260,373, Japanese Patent Application Laid-Open No. 2010-133733, and Japanese Patent Application Laid-Open No. 2010-133734.
  • the linker introduction reaction and the ligand fixation reaction to the porous particles are preferably carried out in a buffer with a pH of 7 to 14.
  • the reaction time for the linker introduction reaction is not particularly limited, but is typically about 0.5 to 72 hours.
  • the reaction temperature may be selected appropriately below the boiling point of the solvent, but is typically about 2 to 100°C.
  • the reaction time for the ligand fixation reaction is not particularly limited, but is typically about 0.1 to 72 hours.
  • the reaction temperature may be selected appropriately below the boiling point of the solvent, but is typically about 2 to 100°C.
  • ligands may be immobilized on porous particles using methods such as those for controlling the orientation of the ligand (U.S. Patent No. 6,399,750; Ljungquist C et al., Eur. J. Biochem., 1989, 186:557-561) or those for accumulating ligands on porous particles using associative groups (JP 2011-256176 A).
  • the amount of ligand immobilized on the porous particles is preferably 10 mg or more, more preferably 25 mg or more, per gram of dry weight of the porous particles, and is preferably 300 mg or less, more preferably 150 mg or less.
  • the hydrophilization reaction may be carried out in the presence of a basic catalyst.
  • basic catalysts include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, and diisopropylethylamine, and these can be used alone or in combination of two or more.
  • the reaction time is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours.
  • the reaction temperature may be selected as appropriate as long as it is below the boiling point of the solvent, but is usually about 2 to 100°C.
  • the resulting ligand-immobilized porous particles can be purified by separation means such as filtration or washing.
  • the ligand-immobilized porous particles may also be classified.
  • the ligand-immobilized porous particles used in the present invention have an average pore size adjusted to fall within a specific range, thereby improving the ability to purify Fc fusion proteins.
  • the average pore size of the ligand-immobilized porous particles is preferably 75 nm or more, more preferably 101 nm or more, and is preferably 120 nm or less, more preferably 110 nm or less.
  • the ligand-immobilized porous particles have an average pore size within the above range, which improves DBC for Fc fusion proteins and pressure resistance during liquid flow.
  • the elution volumes of pullulan, dextran, and sodium chloride can be measured by passing a 20 mM sodium phosphate/150 mM sodium chloride aqueous solution containing pullulan, a 20 mM sodium phosphate/150 mM sodium chloride aqueous solution containing dextran, and a 500 mM sodium chloride aqueous solution through a column packed with the ligand-immobilized porous particles.
  • the porosity of the ligand-immobilized porous particles in a wet state is preferably 70 to 95% (v/v), more preferably 75 to 85% (v/v).
  • the porosity of the ligand-immobilized porous particles in a wet state refers to the ratio of the pore volume (volume of the pores) to the particle volume (the total volume of the non-pore portions of the porous particles, the pore portions of the porous particles, and the ligand portion) of the ligand-immobilized porous particles measured in a wet state.
  • the porosity can be determined based on the elution volume of a 500 mM sodium chloride aqueous solution from a column packed with the ligand-immobilized porous particles and the elution volume of a 20 mM sodium phosphate/150 mM sodium chloride aqueous solution containing dextran from the same column.
  • the average pore diameter and porosity in a wet state of the ligand-immobilized porous particles can be measured according to the method described in the Examples below.
  • the ligand-immobilized porous particles When used to purify Fc fusion proteins, the ligand-immobilized porous particles can demonstrate high performance in terms of high DBC and low non-specific binding (contamination of impurities). Considering that high performance was not achieved when conventional supports were used to purify Fc fusion proteins, this demonstrates that the chromatography support of the present invention has a high ability to purify Fc fusion proteins. Therefore, the ligand-immobilized porous particles can be used as a chromatography support for purifying Fc fusion proteins.
  • a chromatography column is provided, characterized by comprising the chromatography support of the present invention.
  • the chromatography column of the present invention is similar to a conventional chromatography column except that it comprises the chromatography support of the present invention.
  • the chromatography column includes a column container and the chromatography support of the present invention packed in the column container.
  • the chromatography column of the present invention is suitable for use in affinity chromatography.
  • the present invention provides a method for purifying an Fc-fusion protein using the aforementioned chromatography support of the present invention.
  • the method for purifying an Fc-fusion protein according to the present invention can be carried out according to a general procedure for purifying an antibody by affinity chromatography, except that the chromatography support of the present invention is used.
  • the method for purifying an Fc fusion protein according to the present invention comprises the steps of passing a solution containing the Fc fusion protein through a column containing the chromatography support of the present invention to bind the Fc fusion protein to the support (step 1); washing the support (step 2); and recovering the Fc fusion protein from the support (step 3).
  • a solution (sample solution) containing the Fc fusion protein is passed through a column containing the chromatography carrier under conditions that allow the Fc fusion protein to bind to the protein ligand of the carrier.
  • the sample solution include an extract from a culture of cells expressing the Fc fusion protein, or a buffer solution containing the extract.
  • Conditions that allow the Fc fusion protein to bind include, for example, a protein concentration of 0.1 to 10 g/L, a solution pH of 5 to 9, a column residence time of 0.5 to 50 minutes, and a temperature of 0 to 40°C.
  • step 1 most of the substances in the sample solution other than the Fc fusion protein pass through the column.
  • step 2 impurities remaining in the column (e.g., proteins in the sample solution other than the Fc fusion protein) are removed by washing.
  • the packing material can be washed with a neutral buffer solution containing a salt such as NaCl, such as a sodium phosphate/sodium chloride solution, sodium dihydrogen phosphate/disodium hydrogen phosphate solution, citric acid/disodium hydrogen phosphate solution, hydrochloric acid/tris(hydroxymethyl)aminomethane solution, or HEPES/sodium hydroxide solution.
  • a salt such as NaCl
  • an eluent is passed through the column to elute the Fc fusion protein from the column, thereby recovering the purified Fc fusion protein.
  • the eluent can be a buffer solution of pH 2 to 5, such as a citric acid/sodium citrate solution, an acetic acid/sodium acetate solution, or a hydrochloric acid/glycine solution.
  • the type of Fc fusion protein targeted in the Fc fusion protein purification method of the present invention is not particularly limited, but one with a molecular weight of 160 to 300 kDa is preferred.
  • Preferred examples of target Fc fusion proteins include etanercept, rilonacept, and efmoroctocog alfa, with etanercept being more preferred.
  • target Fc fusion proteins include biosimilars of etanercept, rilonacept, or efmoroctocog alfa, such as Fc fusion proteins that share 85% or more amino acid sequence identity with etanercept, rilonacept, or efmoroctocog alfa and whose region corresponding to the Fc fragment has binding ability to the ligand contained in the chromatography support of the present invention.
  • the amino acid sequence of etanercept can be represented by SEQ ID NO: 4.
  • the amino acid sequence of rilonacept can be represented by SEQ ID NO: 5. Therefore, preferred examples of target Fc fusion proteins include Fc fusion proteins consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having 85% or more identity to either of these sequences.
  • PrA-1 and PrA-2 are Fc-binding proteins comprising a homopentamer in which Fc-binding domains are linked in tandem.
  • the Fc-binding domains contained in PrA-1 and PrA-2 are modified domains of the C domain of Protein A (SEQ ID NO: 1), and each consists of an amino acid sequence in which the mutations listed in Table 1 have been introduced into the parent domain of SEQ ID NO: 1.
  • PrA-1 and PrA-2 were expressed and purified as follows. Escherichia coli BL21(DE3) was transformed with the plasmids encoding PrA-1 and PrA-2, respectively, and the resulting transformants were cultured in a rich medium at 37°C until logarithmic growth phase. Subsequently, isopropyl- ⁇ -thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 1 mM, and the transformants were further cultured at 37°C for 4 hours to express the target proteins. The culture medium was then centrifuged, and the supernatant was removed.
  • Escherichia coli BL21(DE3) was transformed with the plasmids encoding PrA-1 and PrA-2, respectively, and the resulting transformants were cultured in a rich medium at 37°C until logarithmic growth phase. Subsequently, isopropyl- ⁇ -thiogalactopyranoside (Wako
  • the resulting cells were disrupted by adding 30 mM Tris buffer (pH 9.5) containing egg white lysozyme (Wako Pure Chemical Industries, Ltd.) and polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical Industries, Ltd.).
  • the recombinant Fc-binding protein was purified from the resulting cell lysate using cation exchange chromatography (SP-Sepharose FF, GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharose FF, GE Healthcare Biosciences).
  • the purified Fc-binding protein was dialyzed against 10 mM citrate buffer, pH 6.0.
  • the purity of the recombinant Fc-binding protein confirmed by SDS-PAGE was 95% or higher.
  • Example 1 Production of Chromatography Support and Purification of Fc Fusion Protein (1) Preparation of Porous Particles Step (a): 2.69 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the polyvinyl alcohol was dissolved by heating and stirring to prepare aqueous solution S. 2.94 g of ethyl cellulose (ETHOCEL TM 14, manufactured by The Dow Chemical Company) was added to 26.44 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.), and the ethyl cellulose was dissolved by heating and stirring to prepare organic solution P.
  • PVA-217 polyvinyl alcohol
  • ETHOCEL TM 14 manufactured by The Dow Chemical Company
  • a monomer composition consisting of 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Inc.), and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.) was dissolved in organic solution P to prepare a monomer solution.
  • 2,2'-azobis(methyl isobutyrate) manufactured by Wako Pure Chemical Industries, Ltd.
  • the resulting reaction solution was filtered to recover the particles.
  • a buffer was obtained by mixing 8.8 g of pure water, 0.1 g of sodium sulfate (Wako Pure Chemical Industries, Ltd.), and 0.03 g of sodium hydroxide (Wako Pure Chemical Industries, Ltd.), and 4.5 g of thioglycerol (Tokyo Chemical Industry Co., Ltd.) was added to prepare a hydrophilization reaction solution.
  • the hydrophilization reaction solution was added to the particles, and the particles were shaken and stirred at 23°C for 16 hours to carry out a hydrophilization reaction.
  • the particles were then dispersed in pure water to a particle concentration of 50% by volume, yielding a ligand-immobilized porous particle dispersion.
  • the average pore diameter of the ligand-immobilized porous particles contained in this dispersion was calculated using the following procedure. This ligand-immobilized porous particle is referred to as "carrier 1.”
  • volume average particle size of the carrier 1 was measured using a laser diffraction/scattering particle size distribution analyzer (LS13320 manufactured by Beckman Coulter, Inc.) in accordance with JIS Z 8825 (2013).
  • the refractive index of the solvent (water) was set to 1.333, and the refractive index of the ligand-immobilized porous particles was set to 1.50.
  • the porosity of carrier 1 in a wet state was measured by the following method.
  • Carrier 1 was packed into a 4 mL volume (1) column (5 mm diameter x 200 mm length) using a 20 mM sodium phosphate/150 mM sodium chloride aqueous solution. 50 ⁇ L of a 500 mM sodium chloride aqueous solution was loaded into the column, and the elution volume (2) was measured.
  • the value obtained by subtracting the elution volume (3) of the dextran-containing aqueous solution (particle void volume) from the column volume was defined as the "particle volume,” and the value obtained by subtracting the elution volume (3) of the dextran from the elution volume of the sodium chloride aqueous solution (2) was defined as the "pore volume.”
  • the ratio (%) of the pore volume to the particle volume was calculated as the "porosity in a wet state" according to the following formula.
  • volume average particle size, porosity, and average pore size were also calculated for Carriers 2 to 8 and Carrier 01, described below, using the same procedures as above.
  • Fc fusion protein Purification of Fc fusion protein by chromatography
  • the Fc fusion protein was purified by chromatography using the carrier 1.
  • a Tricorn 50/200 column manufactured by Cytiva was packed with 4 mL of the carrier 1, and this column was connected to an AKTA school 25 manufactured by Cytiva.
  • a culture solution of cells expressing the Fc fusion protein (etanercept) (etanercept concentration: 1.1 mg/mL) was prepared as a sample solution.
  • the following steps (i) to (iv) constitute one cycle, and this cycle was repeated 100 times.
  • Example 2 Porous particles (referred to as “porous particles 2") were prepared in the same manner as in Example 1(1), except that the ethyl cellulose contained in the organic solution P (ETHOCEL TM 14 manufactured by The Dow Chemical Company) was replaced with ethyl cellulose (ETHOCELTM 10 manufactured by The Dow Chemical Company).
  • ETHOCELTM 14 manufactured by The Dow Chemical Company
  • ETHOCELTM 10 manufactured by The Dow Chemical Company
  • carrier 2 ligand-immobilized porous particles
  • Fc fusion protein purification was carried out in the same manner as in Example 1(3).
  • Example 3 Porous particles (referred to as “porous particles 3") were prepared in the same manner as in Example 1(1), except that the ethyl cellulose contained in the organic solution P (ETHOCEL TM 14 manufactured by The Dow Chemical Company) was replaced with ethyl cellulose (ETHOCEL TM 20 manufactured by The Dow Chemical Company).
  • ETHOCEL TM 14 manufactured by The Dow Chemical Company
  • ETHOCEL TM 20 manufactured by The Dow Chemical Company
  • carrier 3 ligand-immobilized porous particles
  • Fc fusion protein purification was carried out in the same manner as in Example 1(3).
  • Example 4 Porous particles (referred to as “porous particles 4”) were prepared in the same manner as in Example 1(1), except that the stirring speed in step (b) was changed to 490 rpm. Using porous particles 4, ligand-immobilized porous particles (referred to as “carrier 4") were prepared in the same manner as in Example 1(2). Using carrier 4, Fc fusion protein purification was carried out in the same manner as in Example 1(3).
  • Example 5 Porous particles (referred to as “porous particles 5”) were prepared in the same manner as in Example 1(1), except that the stirring speed in step (b) was changed to 230 rpm. Using porous particles 5, ligand-immobilized porous particles (referred to as “carrier 5”) were prepared in the same manner as in Example 1(2). Using carrier 5, Fc fusion protein purification was carried out in the same manner as in Example 1(3).
  • Example 6 Porous particles (referred to as "porous particles 6") were prepared in the same manner as in Example 1(1), except that the amounts of the reagents were changed as follows: in step (a), 23.70 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.), 2.64 g of ethyl cellulose (ETHOCEL TM 14 manufactured by The Dow Chemical Company), 4.35 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.), 0.43 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Ltd.), and 16.94 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.); and in step (b), 1.60 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.), 103.39 g of thiogly
  • Example 7 Porous particles (referred to as "porous particles 7") were prepared in the same manner as in Example 1(1), except that the amounts of the reagents were changed as follows: in step (a), 2-octanone (manufactured by Toyo Gosei Co., Ltd.) was 28.37 g, ethyl cellulose (ETHOCEL TM 14 manufactured by The Dow Chemical Company) was 3.15 g, divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.) was 3.12 g, 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Ltd.) was 0.31 g, and glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.) was 12.17 g; and in step (b), 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was 1.15 g, thioglyce
  • Example 8 Ligand-immobilized porous particles (referred to as “carrier 8") were prepared from the porous particles 1 in the same manner as in Examples 1(1) and 1(2), except that the ligand was changed to PrA-2. Using carrier 8, Fc fusion protein was purified in the same manner as in Example 1(3).
  • Example 9 The Fc fusion protein (rilonacept) was purified using the carrier 1 in the same manner as in Example 1(3), except that the sample solution was changed to a culture medium of rilonacept-expressing cells (rilonacept concentration: 1.1 mg/mL) and, in step (ii), 29.09 mL of the sample solution was loaded onto the column with a retention time of 4 minutes.
  • Example 10 The Fc fusion protein (efmoroctocog alfa) was purified using carrier 1 in the same manner as in Example 1(3), except that the sample solution was changed to a culture medium of efmoroctocog alfa-expressing cells (efmoroctocog alfa concentration: 1.1 mg/mL) and that in step (ii), 22.59 mL of the sample solution was loaded onto the column with a retention time of 4 minutes.
  • a culture medium of efmoroctocog alfa-expressing cells efmoroctocog alfa concentration: 1.1 mg/mL
  • Example 11 The Fc fusion protein was purified using the carrier 1 in the same manner as in Example 1(3), except that the washing solution used in the step (ii) was changed to a 20 mM sodium phosphate/150 mM sodium chloride aqueous solution (pH 7.5).
  • Example 12 The Fc fusion protein was purified using the carrier 1 in the same manner as in Example 1(3), except that the washing solution used in the step (ii) was changed to a 20 mM sodium phosphate/500 mM sodium chloride aqueous solution (pH 6.5).
  • Example 13 The Fc fusion protein was purified using the carrier 1 in the same manner as in Example 1(3), except that the alkaline solution used in the step (iv) was changed to a 0.5 M NaOH aqueous solution.
  • Test Example 1 Measurement of dynamic binding capacity (DBC) The DBC for the target protein was measured at the first and 100th cycles of chromatography in Examples 1 to 13 and Comparative Example 1. That is, the DBC was calculated from the amount of target protein captured at 10% breakthrough at the elution front and the column packed volume. The DBC (mg/mL) at the first cycle and the percentage of DBC at the 100th cycle relative to the first cycle (% DBC) were calculated. The results are shown in Tables 2 and 3.
  • DBC dynamic binding capacity

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Abstract

La présente invention concerne un support chromatographique pour purifier une protéine de fusion Fc et un procédé de purification de protéine de fusion Fc utilisant ledit support chromatographique. Le support chromatographique comprend des particules poreuses auxquelles un ligand est immobilisé, les particules poreuses étant des particules poreuses à base de polymère synthétique ou à base de polymère naturel ; le ligand est au moins une substance choisie dans le groupe constitué par la protéine A, la protéine G, la protéine L et des substances apparentées de celles-ci ; et le diamètre moyen des pores des particules poreuses auxquelles le ligand est immobilisé est de 75 à 120 nm. Le procédé de purification de protéine de fusion Fc comprend : (étape 1) une étape qui consiste à faire passer une solution contenant une protéine de fusion Fc à travers une colonne qui est pourvue dudit support chromatographique et à amener la protéine de fusion Fc à se lier au support ; (étape 2) une étape qui consiste à laver le support ; et (étape 3) une étape qui consiste à collecter la protéine de fusion Fc à partir du support.
PCT/JP2024/041646 2024-02-13 2024-11-25 Support chromatographique pour purifier une protéine de fusion fc et procédé de purification de protéine de fusion fc l'utilisant Pending WO2025173342A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011125673A1 (fr) * 2010-03-31 2011-10-13 Jsr株式会社 Charge pour chromatographie d'affinité
WO2013062105A1 (fr) * 2011-10-28 2013-05-02 Ａｇｃエスアイテック株式会社 Corps sphérique à base de silice et support d'affinité
JP2017524740A (ja) * 2014-07-26 2017-08-31 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 二重特異性抗体のための精製プラットフォーム
WO2022202466A1 (fr) * 2021-03-25 2022-09-29 Jsr株式会社 Procédé de production d'un vecteur chromatographique, procédé de production d'une colonne de chromatographie et vecteur chromatographique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011125673A1 (fr) * 2010-03-31 2011-10-13 Jsr株式会社 Charge pour chromatographie d'affinité
WO2013062105A1 (fr) * 2011-10-28 2013-05-02 Ａｇｃエスアイテック株式会社 Corps sphérique à base de silice et support d'affinité
JP2017524740A (ja) * 2014-07-26 2017-08-31 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 二重特異性抗体のための精製プラットフォーム
WO2022202466A1 (fr) * 2021-03-25 2022-09-29 Jsr株式会社 Procédé de production d'un vecteur chromatographique, procédé de production d'une colonne de chromatographie et vecteur chromatographique

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TUGCU, NIHAL ET AL.: "Maximizing productivity of chromatography steps for purification of monoclonal antibodies", BIOTECHNOLOGY AND BIOENGINEERING, vol. 99, no. 3, 6 August 2007 (2007-08-06), pages 599 - 613, XP071113891, DOI: 10.1002/bit.21604 *
TUSTIAN, ANDREW D. ET AL.: "Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity", MABS, vol. 8, no. 4, 10 March 2016 (2016-03-10), pages 828 - 838, XP055411787, DOI: 10.1080/19420862.2016.1160192 *

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