WO2015199196A1 - アフィニティークロマトグラフィー用担体 - Google Patents
アフィニティークロマトグラフィー用担体 Download PDFInfo
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- WO2015199196A1 WO2015199196A1 PCT/JP2015/068403 JP2015068403W WO2015199196A1 WO 2015199196 A1 WO2015199196 A1 WO 2015199196A1 JP 2015068403 W JP2015068403 W JP 2015068403W WO 2015199196 A1 WO2015199196 A1 WO 2015199196A1
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- group
- affinity chromatography
- carrier
- protein
- mass
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- 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
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
-
- 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
-
- 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
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- 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
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- 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
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
- B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Definitions
- the present invention relates to a carrier for affinity chromatography.
- the present invention relates to a carrier for affinity chromatography useful for purification of proteins such as antibodies.
- Affinity chromatography plays an important role in the research, development and production of proteins including monoclonal antibodies.
- a carrier for affinity chromatography generally includes a solid phase carrier having a ligand that selectively binds to a target substance.
- Affinity chromatography is superior to other chromatography methods such as ion chromatography, gel filtration chromatography, and reverse phase liquid chromatography because the ligand on the solid support has high selectivity for the target substance.
- Economical purification can be achieved at a high yield with a high yield.
- the performance required for affinity chromatography for protein purification includes (1) non-specific adsorption of impurities other than the target protein, and (2) binding capacity does not decrease due to long-term storage, resulting in storage stability. And (3) sufficient mechanical strength for column operation.
- agarose particles Patent Documents 1 and 2
- porous particles made of a copolymer of styrene-divinylbenzene Patent Documents 3 and 4
- methacrylate vinyl monomer as a solid phase carrier for affinity chromatography support
- Porous particles Patent Documents 5 and 6) made of a solid polymer
- agarose particles since agarose particles generally have a low elastic modulus, they have the disadvantage that the column pressure increases when a medium is flowed at a high speed. Moreover, since agarose particles are generally produced from natural seaweed through a long and complicated process, it is difficult to obtain particles of a certain quality.
- porous particles made of a copolymer of an aromatic vinyl monomer such as styrene-divinylbenzene are generally excellent in alkali resistance, but have low hydrophilicity, and therefore have low ligand activity, and are resistant to target substances. Has the disadvantage of low mechanical coupling capacity.
- the porous particles made of a polymer of a methacrylate vinyl monomer have high hydrophilicity and mechanical strength, further improvement in the ability to remove impurities has been desired.
- the problem to be solved by the present invention is to provide a carrier for affinity chromatography that is excellent in storage stability in which non-specific adsorption of impurities is suppressed and a decrease in dynamic binding capacity due to long-term storage is suppressed. .
- a solid phase carrier containing a copolymer containing specific amounts of structural units derived from an epoxy group-containing monovinyl monomer and structural units derived from a polyvinyl monomer A specific affinity chromatography carrier having a ligand that binds to the solid phase carrier and a ring-opened epoxy group is found to have a high dynamic binding capacity of the target substance, and is excellent in impurity removal ability and storage stability, The present invention has been completed.
- the present invention relates to ⁇ 1> (M-1) a structural unit derived from an epoxy group-containing monovinyl monomer: more than 20 parts by mass and not more than 99.5 parts by mass with respect to all structural units; 2) Structural unit derived from a polyvinyl monomer: a solid phase carrier containing a copolymer containing 0.5 to 80 parts by mass with respect to all structural units; A ring-opened epoxy group obtained by ring-opening an epoxy group; A ligand for affinity chromatography comprising a ligand bound to the solid phase carrier,
- the above column for affinity chromatography is packed in an arbitrary column container, the column is replaced with 20 mM sodium phosphate buffer (pH 7.5) containing 2 M NaCl, and the pH in the column is left at 40 ° C. for 7 days.
- the rising width is 2 or less,
- a carrier for affinity chromatography is provided.
- the present invention also provides an affinity chromatography column in which a column container is filled with ⁇ 2> the carrier for affinity chromatography described in ⁇ 1> above.
- the present invention provides a method for purifying a target substance, characterized by using ⁇ 3> the carrier for affinity chromatography described in ⁇ 1> above.
- HCP Host Cell Protein
- the solid phase carrier constituting the affinity chromatography carrier of the present invention is: (M-1) Structural unit derived from epoxy group-containing monovinyl monomer: more than 20 parts by mass and not more than 99.5 parts by mass with respect to all structural units, and (M-2) derived from polyvinyl monomer Structural unit: containing a copolymer containing 0.5 to 80 parts by mass with respect to all structural units, In addition, it includes a ring-opened epoxy group obtained by opening an epoxy group.
- the form of the solid phase carrier may be any of monolith, membrane, hollow fiber, particle, cassette, chip and the like, but particle is preferred.
- the solid phase carrier is preferably a porous material such as porous particles from the viewpoint of improving the surface area.
- porous particles porous polymer particles are preferable.
- the solid phase carrier may contain a natural polymer or a synthetic polymer in addition to the copolymer.
- the epoxy group-containing monovinyl monomer is a monomer having one polymerizable vinyl group (a group having an ethylenically unsaturated bond) and one or more epoxy groups in one molecule.
- the epoxy group-containing vinyl monomer can be used to introduce an appropriate amount of an epoxy group into a solid phase carrier to obtain an appropriate amount of ligand binding or an amount of ring-opened epoxy group.
- epoxy group-containing monovinyl monomer examples include glycidyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth) acrylate, ⁇ - (meth) acryl- ⁇ - Hydroxy group-free (meth) acrylic esters such as glycidyl polyethylene glycol; Hydroxy group-containing (meth) acrylic esters such as glycerin mono (meth) acrylate glycidyl ether; Aromatic monovinyl compounds such as vinylbenzyl glycidyl ether Allyl glycidyl ether, 3,4-epoxy-1-butene, 3,4-epoxy-3-methyl-1-butene, and the like.
- the epoxy group-containing monovinyl monomer is preferably an epoxy group-containing (meth) acrylic acid ester or an epoxy group-containing aromatic monovinyl compound from the viewpoint of antifouling property or storage stability.
- (Meth) acrylic acid esters are more preferred, glycidyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate glycidyl ether is more preferred, and glycidyl (meth) acrylate is particularly preferred.
- the epoxy group-containing monovinyl monomer is preferably a water-insoluble one because the amount of ring-opened epoxy groups can be easily controlled.
- water-insoluble means that 10 g or more does not dissolve in 100 mL of water at room temperature (25 ° C.).
- the epoxy group-containing monovinyl monomer may be a commercially available product, or may be synthesized according to a known method.
- the content of the structural unit derived from the epoxy group-containing monovinyl monomer is more than 20 parts by mass and 99.5 parts by mass or less with respect to all the structural units.
- the content is 20 parts by mass or less, the amount of the ring-opened epoxy group is reduced, the hydrophilicity of the support for affinity chromatography after ligand binding is lowered, and the ability to remove impurities is lowered.
- it exceeds 99.5 parts by mass the mechanical strength becomes low, and it becomes impossible to withstand handling as a carrier for affinity chromatography.
- the content of the structural unit derived from the epoxy group-containing monovinyl monomer is preferably 25 parts by mass or more, more preferably 30 parts by mass or more, with respect to all the structural units, from the viewpoints of antifouling property and storage stability. More preferably, it is 40 parts by mass or more, particularly preferably 50 parts by mass or more, and from the viewpoint of antifouling property, storage stability, etc., preferably 95 parts by mass or less, more preferably 90 parts by mass with respect to all structural units. Part or less, more preferably 85 parts by weight or less, particularly preferably 80 parts by weight or less.
- the polyvinyl monomer is a vinyl monomer having two or more polymerizable vinyl groups (groups having an ethylenically unsaturated bond) in one molecule.
- the polyvinyl monomer will be described by being divided into a hydroxy group-free polyvinyl monomer and a hydroxy group-containing polyvinyl monomer.
- a polyvinyl monomer can be used individually by 1 type or in combination of 2 or more types.
- non-hydroxy group-containing polyvinyl monomer examples include ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, propylene glycol di ( (Meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di (meth) acrylate, 1,4-butanediol di (meth) acrylate, 1,6-hexanediol di (Meth) acrylates such as (meth) acrylate, trimethylolpropane tri (meth) acrylate, pentaerythritol tetra (meth) acrylate; divinylbenzene, tri Aromatic polyvinyl compounds such as Nirubenzen; butadiene, isocyanuric acid diallyl
- hydroxy group-free polyvinyl monomer (meth) acrylic acid esters and aromatic polyvinyl compounds are preferable.
- the number of polymerizable vinyl groups contained in the hydroxy group-free polyvinyl monomer is preferably 2 to 5 and more preferably 2 or 3 in one molecule.
- hydroxy group-containing polyvinyl monomer (meth) acrylic acid esters of polyhydric alcohols, (meth) acrylic acid esters of two or more substituted saccharides, and (meth) acrylamides of polyhydric alcohols are preferable.
- (Meth) acrylic acid esters of polyhydric alcohol include glycerin di (meth) acrylate, trimethylolethane di (meth) acrylate, trimethylolpropane di (meth) acrylate, butanetriol di (meth) acrylate, pentaerythritol di (Meth) acrylate, pentaerythritol tri (meth) acrylate, dipentaerythritol di (meth) acrylate, dipentaerythritol tri (meth) acrylate, dipentaerythritol tetra (meth) acrylate, dipentaerythritol penta (meth) acrylate, inositol Examples include di (meth) acrylate, inositol tri (meth) acrylate, and inositol tetra (meth) acrylate.
- Examples of (meth) acrylic acid esters having two or more substitutions of various sugars include, for example, glucose di (meth) acrylate, glucose tri (meth) acrylate, glucose tetra (meth) acrylate, mannitol di (meth) acrylate, mannitol tri ( Examples include meth) acrylate, mannitol tetra (meth) acrylate, and mannitol penta (meth) acrylate.
- examples of the hydroxy group-containing polyvinyl monomer include, in addition to those exemplified above, dehydration condensation reaction products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth) acrylic acid. it can.
- the number of polymerizable vinyl groups contained in the hydroxy group-containing polyvinyl monomer is preferably 2 to 5 and more preferably 2 or 3 in one molecule.
- polyvinyl monomer good porosity and mechanical strength can be obtained with a relatively small amount of use, and from the viewpoint of limiting the use amount of an epoxy group-containing monovinyl monomer, Hydroxy group-free polyvinyl monomers are preferred.
- non-hydroxy group-containing polyvinyl monomers from the viewpoint of antifouling properties and storage stability, (meth) acrylic acid esters having 3 polymerizable vinyl groups, 2 or 2 polymerizable vinyl groups Three aromatic polyvinyl compounds are particularly preferred. Particularly preferred specific examples include trimethylolpropane tri (meth) acrylate, divinylbenzene, and trivinylbenzene.
- the content of the structural unit derived from the polyvinyl monomer is 0.5 to 80 parts by mass with respect to the total structural unit.
- the content of the structural unit derived from the polyvinyl monomer is less than 0.5 parts by mass, the mechanical strength of the solid phase carrier becomes low, and it cannot be handled as a carrier for affinity chromatography.
- the hydrophilicity is lowered, the effect of reducing HCP cannot be obtained, and the activity of the ligand is lowered.
- the content of the structural unit derived from the polyvinyl monomer is preferably at least 5 parts by mass, more preferably at least 10 parts by mass, and even more preferably from the viewpoint of antifouling properties and storage stability.
- (M-3) Structural unit derived from monovinyl monomer not containing epoxy group examples of the copolymer contained in the solid phase carrier used in the present invention include (M-3) a monovinyl monomer not containing an epoxy group in addition to the structural unit (M-1) and the structural unit (M-2).
- the structural unit derived from is preferably one further containing 40 parts by mass or less based on the total structural unit.
- the monovinyl monomer not containing an epoxy group is a vinyl monomer having one polymerizable vinyl group (group having an ethylenically unsaturated bond) in one molecule and no epoxy group.
- the monovinyl monomer monomer that does not contain an epoxy group will be described by being divided into a hydroxy group-free monovinyl monomer that does not contain an epoxy group and a hydroxy group-containing monovinyl monomer that does not contain an epoxy group.
- a nonionic monomer is preferable from the viewpoint of preventing nonspecific adsorption of impurities during purification.
- nonionic monomers include (meth) acrylic such as methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, cyclohexyl (meth) acrylate, and methoxyethyl (meth) acrylate.
- Acid esters such as (meth) acrylamide, dimethyl (meth) acrylamide, (meth) acryloylmorpholine, diacetone (meth) acrylamide, etc., may be used alone or in combination of two or more Can be used.
- Nonionic monomers include hydrophobic (meth) acrylates such as 2-ethylhexyl (meth) acrylate and stearyl (meth) acrylate; aromatic vinyl such as styrene, ⁇ -methylstyrene and ethylvinylbenzene.
- a compound may be used, but from the viewpoint of suppressing non-specific adsorption of impurities during purification, it is not such a monomer, but among the monomers exemplified above, an alkyl group having 5 or less carbon atoms or alkoxyalkyl (Meth) acrylic acid alkyl esters having a group and the above (meth) acrylamides are preferred.
- hydroxy group-containing monovinyl monomers that do not contain an epoxy group
- examples of hydroxy group-containing monovinyl monomers that do not contain an epoxy group include glycerol mono (meth) acrylate, trimethylolethane mono (meth) acrylate, trimethylolpropane mono (meth) acrylate, butanetriol mono (meth) acrylate, (Meth) acrylics such as pentaerythritol mono (meth) acrylate, dipentaerythritol mono (meth) acrylate, inositol mono (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, and polyethylene glycol (meth) acrylate Acid esters; (meth) acrylamides such as hydroxyethyl (meth) acrylamide can be used, and one kind can be used alone or two or more kinds can be used in combination . Further, the number of hydroxy
- a hydroxy group-containing monovinyl monomer not containing an epoxy group is preferable from the viewpoint of antifouling property, and glycerol mono (meth) acrylate, hydroxyethyl (metaethyl) ) Acrylate, hydroxypropyl (meth) acrylate, and hydroxyethyl (meth) acrylamide are more preferable, glycerol mono (meth) acrylate and hydroxyethyl (meth) acrylamide are more preferable, and hydroxyethyl (meth) acrylamide is particularly preferable.
- the content of the structural unit derived from the monovinyl monomer not containing an epoxy group is preferably 40 parts by mass or less based on the total structural unit.
- the content is 40 parts by mass or less, in the case of a hydroxy group-containing vinyl monomer, the hydrophilicity of the porous particles is improved, aggregation is suppressed, the activity of the ligand is increased, and the movement of the target substance is increased.
- the amount of mechanical binding is improved.
- the mechanical strength is improved.
- the content of the structural unit is more preferably 35 parts by mass or less, further preferably 30 parts by mass or less, still more preferably 25 parts by mass or less, and particularly preferably 20 parts by mass or less with respect to the total structural units.
- 0 mass part may be sufficient as content of the structural unit derived from the monovinyl monomer which does not contain an epoxy group.
- 5 mass parts or more are preferable with respect to all the structural units.
- Preferred combinations of the contents of the structural units (M-1) to (M-3) are: the structural unit (M-1): 40 to 90 parts by mass with respect to all the structural units, and the structural unit (M-2): all 10 to 60 parts by mass with respect to the structural unit, structural unit (M-3): 0 to 25 parts by mass with respect to all structural units, and a more preferred combination is structural unit (M-1): 40 with respect to all structural units.
- structural unit (M-2) 15 to 60 parts by mass with respect to all structural units
- structural unit (M-3) 0 to 25 parts by mass with respect to all structural units
- Structural unit (M-1) 40 to 80 parts by mass for all structural units
- Structural unit (M-2) 20 to 60 parts by mass for all structural units
- Structural unit (M-3) for all structural units The amount is 0 to 20 parts by mass.
- the epoxy group contained in the solid phase carrier before binding to the ligand is a functional group for binding the ligand, and further serves as a functional group for improving hydrophilicity as a carrier for affinity chromatography after ring opening.
- the epoxy group content of the solid phase carrier before ligand binding is preferably 1 to 6 mmol / g, more preferably 2 to 5 mmol / g, and particularly preferably 2.5 to 4 mmol / g.
- the epoxy group content of the solid phase carrier before ligand binding is 6 mmol / g or less, the residual amount of the epoxy group after ligand binding decreases, and the activity of the ligand decreases when stored as a carrier for affinity chromatography. It is suppressed, and the decrease in the dynamic binding capacity of the target substance when used repeatedly is also suppressed.
- the epoxy group content of the solid phase carrier before ligand binding should be adjusted by the amount of the epoxy group-containing monovinyl monomer, the polymerization temperature, the polymerization time, the pH of the polymerization solution, and the ring-opening treatment after polymerization. I can do it.
- a method for producing a solid phase carrier will be described by taking a case where the solid phase carrier is a porous particle as an example.
- the porous particles can be produced by known seed polymerization, suspension polymerization or the like.
- An example of the seed polymerization method is a two-stage swelling polymerization method described in JP-B-57-24369.
- a polymerization initiator, a porous agent, an aqueous medium, a dispersion stabilizer, a surfactant, a polymerization regulator, a polymerization inhibitor, seed particles, and the like are used as necessary.
- a preferred polymerization method in the production of porous particles is a suspension polymerization method of an aqueous mixture containing a monomer mixture and a porosifying agent as essential components.
- a polymerization initiator is dissolved in a mixed solution (monomer solution) containing a monomer mixture and a porosifying agent, and suspended in an aqueous medium.
- Examples thereof include a method, a method in which a mixed solution (monomer solution) containing a monomer mixture and a porous agent is suspended in an aqueous medium and heated to a predetermined temperature, and a polymerization initiator is added for polymerization. .
- radical polymerization initiators include azo initiators, peroxide initiators, redox initiators, and the like. Specific examples include azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2, Examples include 4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, benzoyl peroxide-dimethylaniline, and the like.
- the total amount of the polymerization initiator used is usually about 0.01 to 10 parts by mass with respect to 100 parts by mass of the total amount of monomers.
- the above porous agent is used to produce porous particles, and is present together with the monomer in the polymerization in the oil droplets, and has a role of forming pores as a non-polymerized component.
- the porous agent is not particularly limited as long as it can be easily removed on the porous surface, and examples thereof include linear polymers that are soluble in various organic solvents and monomer mixtures. These may be used in combination.
- the porosifying agent examples include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and undecane; alicyclic hydrocarbons such as cyclohexane and cyclopentane; benzene, toluene, xylene, naphthalene, and ethylbenzene.
- Aromatic hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, chlorobenzene and the like; butanol, pentanol, hexanol, heptanol, hexanol, 4-methyl-2-pentanol, Aliphatic alcohols such as 2-ethyl-1-hexanol; alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, Ketones such as non, 2-octanone and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole and ethoxybenzene; in addition to esters such as isopentyl acetate, butyl acetate, 3-meth
- aqueous medium examples include aqueous water-soluble polymer aqueous solutions, and examples of the water-soluble polymer include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinyl pyrrolidone, starch, and gelatin.
- the total amount of the aqueous medium used is usually about 200 to 7000 parts by mass with respect to 100 parts by mass of the total amount of monomers.
- a dispersion stabilizer such as sodium chloride, sodium sulfate, sodium carbonate, calcium carbonate, calcium phosphate may be used.
- Various surfactants including anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfate ester salts, alkyl phosphate ester salts, and fatty acid salts may also be used.
- a polymerization inhibitor such as nitrite such as sodium nitrite, iodide salt such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, ferric chloride and the like can be used.
- polymerization preparation agents such as dodecyl mercaptan.
- the polymerization temperature may be determined according to the polymerization initiator. For example, when azobisisobutyronitrile is used as the polymerization initiator, it is preferably 50 to 100 ° C., more preferably 60 to 90 ° C.
- the polymerization time is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.
- washing with water and / or hot water in order to remove the water-soluble polymer attached to the obtained porous particles.
- washing with a good solvent for seed particles and / or a porosifying agent is preferable from the viewpoint of easy binding of a ligand described later.
- the washing solvent acetone, ethanol, isopropyl alcohol or the like can be preferably used.
- the carrier for affinity chromatography of the present invention has a ring-opened epoxy group obtained by opening an epoxy group.
- This ring-opened epoxy group opens a residual epoxy group other than the epoxy group contained in the copolymer, that is, the epoxy group bonded to the ligand, after binding the ligand to the solid phase carrier containing the copolymer. It can be obtained by ringing.
- epoxy groups include epoxy groups introduced later by epichlorohydrin, diglycidyl ether compounds, etc., which stage in the production of the affinity chromatography carrier of the present invention It doesn't matter if it was introduced in
- the affinity chromatography carrier of the present invention In the affinity chromatography carrier of the present invention, the epoxy group on the surface of the solid phase carrier is almost ring-opened, and the affinity chromatography carrier is brought into contact with a high concentration of chloride ions to open the remaining epoxy groups. The amount of hydroxide ions released when ringed is extremely small. Therefore, the affinity chromatography carrier of the present invention is packed in an arbitrary column container with the affinity chromatography carrier, and the column is replaced with 20 mM sodium phosphate buffer having a pH of 7.5 containing 2 M NaCl. The pH increase in the column when allowed to stand for 7 days is 2 or less. With such a configuration, the impurity removal capability and storage stability are excellent. The pH increase is preferably 1.9 or less, and more preferably 1.7 or less when further improving the ability to remove impurities. Moreover, the lower limit value of the pH increase range is not particularly limited, but is, for example, 0.01.
- the pH increase range can be obtained from the following formula, and can be measured by the method described in the examples in more detail.
- the pH increase is measured on a new affinity chromatography carrier.
- the new carrier for affinity chromatography means a carrier that has not been used before. For example, if it is used for antibody purification, it means a carrier that has not been used for antibody purification.
- (PH increase range) (Load pH) ⁇ (Initial pH)
- initial pH means a pH value when a pH 7.5 20 mM sodium phosphate buffer containing 2 M NaCl is passed through the column and the pH in the column reaches equilibrium.
- the “load pH” is determined by measuring the initial pH and allowing it to stand in a 40 ° C. incubator for 7 days, and then passing a 20 mM sodium phosphate buffer solution containing 2 M NaCl over pH 7.5 through the column again. It means the value of the pH at which the liquid is liquefied and the pH becomes the highest.
- the ring-opened epoxy group produced by ring-opening of the epoxy group is represented by the following formula (1) from the viewpoint of preventing nonspecific adsorption of impurities and preventing a decrease in the activity of the ligand during storage.
- the group represented is preferred.
- R 1 represents a divalent organic group having 1 to 6 carbon atoms
- R 2 represents a hydrogen atom or a monovalent organic group having 1 to 10 carbon atoms
- X represents a thio group (> S), a sulfinyl group (> S ⁇ O), a sulfonyl group (> S ( ⁇ O) 2 ), an imino group (> NH), or an oxy group (> O), * Indicates a binding position.
- R 1 represents a divalent organic group having 1 to 6 carbon atoms.
- the number of carbon atoms of the organic group is preferably 1 to 4, more preferably 1 or 2.
- the divalent organic group may be linear or branched.
- Such a divalent organic group includes a group having at least one selected from an ether bond, an imino group and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group and a divalent hydrocarbon group.
- a divalent hydrocarbon group is preferable.
- the divalent hydrocarbon group is preferably a divalent aliphatic hydrocarbon group, more preferably an alkanediyl group.
- alkanediyl group examples include methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2 -Diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group, etc. Can be mentioned.
- R 2 represents a hydrogen atom or a monovalent organic group having 1 to 10 carbon atoms, preferably a monovalent organic group having 1 to 10 carbon atoms.
- Such an organic group is preferably an organic group represented by the following formula (2) or (3) from the viewpoint of dynamic binding capacity and antifouling property when a ligand is bound, The organic group represented is more preferred.
- R 3 represents a divalent or trivalent organic group having 1 to 10 carbon atoms, n represents 1 or 2, ** represents a bonding position with X in the formula (1).
- R 4 represents a divalent or trivalent organic group having 1 to 10 carbon atoms
- Y represents an acidic group or an amino group
- m represents 1 or 2
- ** represents a bonding position with X in the formula (1).
- the number of carbon atoms of the divalent or trivalent organic group represented by R 3 in formula (2) or R 4 in formula (3) is from the viewpoint of dynamic binding capacity when a ligand is bound, and the like. 1-8 are preferred, 1-6 are more preferred, and 2-4 are even more preferred.
- examples of the divalent organic group include divalent hydrocarbon groups, which may be linear or branched.
- the divalent hydrocarbon group is preferably a divalent aliphatic hydrocarbon group, more preferably an alkanediyl group.
- the number of carbon atoms of such an alkanediyl group is preferably 1 to 8, more preferably 1 to 6, and further preferably 2 to 4 from the viewpoint of dynamic binding capacity when a ligand is bound.
- alkanediyl group examples include methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1, Examples include 2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group and the like.
- examples of the trivalent organic group include a trivalent hydrocarbon group, which may be linear or branched.
- the trivalent hydrocarbon group is preferably a trivalent aliphatic hydrocarbon group, and more preferably an alkanetriyl group.
- the number of carbon atoms of such an alkanetriyl group is preferably 1 to 8, more preferably 1 to 6, and further preferably 2 to 4 from the viewpoint of dynamic binding capacity and antifouling property when a ligand is bound. .
- alkanetriyl group examples include methane-1,1,1-triyl group, ethane-1,1,2-triyl group, propane-1,2,3-triyl group, propane-1,2, And 2-triyl group.
- n 1 or 2, but 2 is preferable.
- Y represents an acidic group or an amino group, but an acidic group is preferred.
- the acidic group include a carboxy group, a phosphate group, a sulfo group, and salts thereof.
- the salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic ammonium salt and the like.
- m represents 1 or 2, but 1 is preferred.
- X represents a thio group (> S), a sulfinyl group (> S ⁇ O), a sulfonyl group (> S ( ⁇ O) 2 ), an imino group (> NH), or an oxy group (> O).
- a group (> S), a sulfinyl group (> S ⁇ O), and an oxy group (> O) are preferred, a thio group (> S), a sulfinyl group (> S ⁇ O) are more preferred, and a sulfinyl group (> S ⁇ O). Is particularly preferred.
- Examples of the ring opening method of the epoxy group in the solid phase carrier include a method of stirring in an aqueous solvent in the presence of an acid or an alkali. Stirring may be performed by heating or at room temperature.
- hydrophilic groups such as hydroxy group; amino group; carboxy group, phosphoric acid group, sulfo group, these salts, etc. are mentioned.
- the salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic ammonium salt and the like.
- the amount of the acid, alkali or blocking agent used is usually 0.1 to 40 molar equivalents per 1 mol of the remaining epoxy group. Since the remaining epoxy groups are bonded to the ligand at multiple points to reduce the activity of the ligand during storage, blocking is preferably performed in an excess amount of 2 to 40 molar equivalents.
- the reaction time for the ring-opening reaction is not particularly limited, but is usually about 0.5 to 72 hours, preferably 1 to 48 hours.
- the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100 ° C.
- a basic catalyst include triethylamine, N, N-dimethyl-4-aminopyridine, diisopropylethylamine, and the like.
- One kind can be used alone, or two or more kinds can be used in combination.
- a preferred ring-opening epoxy group is a ring-opening epoxy group obtained by ring-opening an epoxy group contained in a solid phase carrier with a mercapto compound and / or a polyhydric alcohol, and is based on a ring-opening group by a blocking agent having an amino group.
- a blocking agent having an amino group.
- non-specific adsorption is reduced and the amount of dynamic binding is increased.
- a thio group can be oxidized to a sulfinyl group using an oxidizing agent to further improve hydrophilicity.
- the oxidizing agent is roughly classified into an organic oxidizing agent and an inorganic oxidizing agent, and examples of the organic oxidizing agent include peracetic acid, perbenzoic acid, and metachloroperbenzoic acid.
- examples of the inorganic oxidizing agent include hydrogen peroxide, chromic acid, permanganate and the like.
- these oxidizing agents can be used individually by 1 type or in combination of 2 or more types.
- the total amount of the oxidizing agent used is usually about 0.1 to 10 molar equivalents, preferably 0.5 to 3 molar equivalents, per 1 mole of thio group.
- the oxidation reaction is preferably performed in the presence of a solvent.
- a solvent examples include water; amide solvents such as dimethylformamide and dimethylacetamide; alcohol solvents such as methanol and ethanol. These solvents can be used alone or in combination of two or more.
- the total amount of the solvent used is usually about 1 to 50 times by mass, preferably 5 to 15 times by mass with respect to the solid phase carrier used as a raw material.
- the reaction time for the oxidation reaction is not particularly limited, but is usually about 1 to 72 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 1 to 90 ° C.
- the carrier for affinity chromatography of the present invention comprises a ligand bound to a solid phase carrier.
- the type of ligand is not particularly limited as long as it has an appropriate affinity for a target substance.
- protein such as protein A, protein G, protein L, Fc-binding protein, and avidin
- peptide such as insulin Antibodies such as monoclonal antibodies
- enzymes such as monoclonal antibodies
- enzymes such as monoclonal antibodies
- enzymes such as monoclonal antibodies
- enzymes such as monoclonal antibodies
- enzymes hormones
- DNA DNA
- RNA carbohydrates such as heparin, Lewis X and ganglioside
- iminodiacetic acid synthetic dyes, 2-aminophenylboronic acid, 4-aminobenzamidine, glutathione, biotin and the like
- Low molecular weight compounds such as derivatives can be used.
- the ligand illustrated above may use the whole, the fragment obtained by a recombinant, an enzyme treatment
- examples of ligands suitable for immunoglobulin separation or purification include immunoglobulin binding proteins.
- the immunoglobulin binding protein is preferably at least one selected from the group consisting of protein A, protein G, protein L, Fc binding protein, and functional variants thereof. Among them, protein A, protein G, and functional variants thereof are preferable, and protein A, and functional variants thereof are more preferable.
- the term “protein” refers to any molecule having a peptide structural unit, for example, a concept including a partial fragment of a natural protein or a variant obtained by artificially modifying the amino acid sequence of a natural protein. is there.
- the “immunoglobulin binding domain” represents a functional unit of a polypeptide having immunoglobulin binding activity alone, and the “immunoglobulin binding protein” has a specific affinity for immunoglobulin, and , Which represents a protein comprising an “immunoglobulin binding domain”.
- “Immunoglobulin binding” refers to binding to a region other than the complementarity determining region (CDR) of an immunoglobulin molecule, particularly to an Fc fragment.
- the term “ligand” used in connection with affinity chromatography refers to a molecule that binds to a target substance for affinity chromatography.
- the epoxy group contained in the solid phase carrier is directly used as a binding site for the ligand because the process is simple.
- the alcoholic hydroxyl group generated by ring-opening the epoxy group contained in the solid phase carrier is activated with a tosyl group and the like, and then the ligand is bound, the epoxy group contained in the solid phase carrier or the epoxy group
- the linker may be further extended from the group generated by ring opening of the group, and then the ligand may be bound to the solid phase carrier via the linker.
- diglycidyl ether compounds such as diglycidyloxyalkane and polyalkylene glycol diglycidyl ether are preferable.
- the ligand binding conditions vary depending on the epoxy group content of the solid phase carrier before ligand binding and the type of ligand, but methods known to those skilled in the art can be employed.
- the ligand is a protein
- the amino group at the N-terminal of the protein, lysine and cysteine contained in the protein can be the reaction site with the epoxy.
- binding proteins for example, using an aqueous solution of buffers close to the isoelectric point of the protein, adding a salt such as sodium chloride or sodium sulfate as necessary, and mixing the protein and the solid support at 0 to 40 ° C.
- the protein as a ligand can be bound by reacting for 1 to 48 hours.
- the binding amount of the ligand is appropriately adjusted depending on the type of ligand, the type of target substance, etc., but when binding an immunoglobulin-binding protein such as protein A as a ligand, it is preferably 10 to 200 mg, more preferably 25 to 100 mg. When binding an immunoglobulin-binding protein such as protein A as a ligand, the binding capacity of the ligand per 1 g of the solid phase carrier is 10 mg or more, and the dynamic binding capacity is excellent. The amount of eluate used for elution of the prepared antibody is particularly suitable.
- the particle size of the porous particle is usually 35 to 100 ⁇ m, preferably 40 to 85 ⁇ m. When the particle size is 35 ⁇ m or more, the pressure characteristics are excellent. When the thickness is 100 ⁇ m or less, a filler having a high dynamic binding capacity can be obtained.
- the particle size of the porous particles can be adjusted according to the polymerization conditions.
- the “particle diameter” in the present invention means a volume average particle diameter obtained by a laser diffraction / scattering particle size distribution analyzer (LS13 320 manufactured by Beckman Coulter, Inc.).
- the specific surface area of the porous particle is a pore size of 10 nm when a pore corresponding to a pore diameter range of 10 to 5000 nm is measured.
- the specific surface area at ⁇ 5000 nm is preferably 70 m 2 / g or more, more preferably 90 m 2 / g or more.
- the “specific surface area” in the present invention is a value obtained by dividing the surface area of pores having a pore diameter of 10 to 5000 nm obtained by a mercury porosimeter (manufactured by Shimadzu Corporation Autopore IV9520) by the dry mass of the particles.
- the carrier for affinity chromatography of the present invention has a high impurity removal ability and excellent storage stability, and is particularly useful for purification of proteins such as antibodies.
- the affinity chromatography column of the present invention is a column container filled with the carrier for affinity chromatography of the present invention.
- the target substance purification method of the present invention is characterized by using the carrier for affinity chromatography of the present invention. Purification is the same as in the conventional method except that the carrier for affinity chromatography of the present invention is used.
- the preparation step for preparing the composition containing the target substance and the composition are passed through the chromatography column of the present invention. Examples of the method include a liquid passing step for liquid and an elution step for eluting the target substance adsorbed on the carrier by the liquid passing step.
- the purification method of the present invention is suitable for protein purification and particularly suitable for immunoglobulin purification.
- Example 1 (1) Add 3.58 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) to 360 g of pure water, dissolve the polyvinyl alcohol by heating and stirring, cool, and then add sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 0 .36 g, sodium sulfate (manufactured by Wako Pure Chemical Industries) 0.36 g, and sodium nitrite (manufactured by Wako Pure Chemical Industries) 0.18 g were added and stirred to prepare an aqueous solution S.
- PVA-217 manufactured by Kuraray Co., Ltd.
- a monomer composition composed of 12.00 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.) and 1.33 g of divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.) is dissolved in 24.43 g of diisobutyl ketone (manufactured by Mitsui Chemicals, Inc.).
- a monomer solution was prepared.
- the entire amount of the aqueous solution S was put into a 500 mL separable flask, a thermometer, a stirring blade and a cooling pipe were attached, set in a hot water bath, and stirring was started in a nitrogen atmosphere.
- the whole amount of the monomer solution was put into a separable flask and heated by a hot water bath.
- 2,2′-azoisobutyronitrile manufactured by Wako Pure Chemical Industries, Ltd.
- 0 .53 g was added and the temperature was maintained at 86 ° C.
- stirring was performed for 3 hours.
- the reaction solution was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles.
- the particles were dispersed in pure water so that the concentration of the particles was 10% by mass, thereby obtaining a porous particle dispersion.
- Example 2 In step (1) of Example 1, 9.20 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.), 0.58 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.) 1 A monomer composition consisting of .73 g was dissolved in a mixed solution of 19.59 g of diisobutyl ketone (Mitsui Chemicals) and 6.05 g of anisole (Wako Pure Chemical Industries) to prepare a monomer solution. The same operations as in steps (1) and (2) of Example 1 were performed to obtain a porous particle dispersion.
- protein A was immobilized by the same operation as in step (3) of Example 1 to produce protein A-immobilized particles.
- the produced protein A fixed particles are dispersed in 40 mL of 1.0 M 2-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M sodium sulfate (pH 8.3), and are shaken and stirred at 25 ° C. for 17 hours. This opened the unreacted epoxy group.
- the obtained protein A-fixed particles in which the unreacted epoxy group was opened were mixed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M sodium hydroxide aqueous solution, 0.1 M sodium citrate buffer (pH 3). . 2) to obtain a packing material 2 for affinity chromatography.
- Example 3 In step (1) of Example 1, 8.18 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.), 1.36 g of N- (2-hydroxyethyl) acrylamide (manufactured by Tokyo Chemical Industry Co., Ltd.) and trimethylolpropane trimethacrylate (Sartomer) The monomer composition consisting of 4.09 g was dissolved in a mixed solution of 21.97 g of 4-methyl-2-pentanol (manufactured by Wako Pure Chemical Industries) and 2.95 g of diethyl carbonate (manufactured by Wako Pure Chemical Industries).
- the same operations as in steps (1) and (2) of Example 1 were performed to obtain a porous particle dispersion.
- protein A was immobilized by the same operation as in step (3) of Example 1 to produce protein A-immobilized particles.
- the produced protein A fixed particles (PA-1) are dispersed in 40 mL of 1.0 M 1-thioglycerol (manufactured by Asahi Chemical Industry Co., Ltd.) / 0.1 M sodium sulfate (pH 8.3), and the mixture is dispersed at 25 ° C. for 17 hours.
- the unreacted epoxy group was opened by shaking and stirring.
- the obtained protein A-fixed particles (PA-2) in which the unreacted epoxy group was opened were mixed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M aqueous sodium hydroxide solution, 0.1 M citric acid. Washing with sodium acid buffer (pH 3.2) gave a packing material 3 for affinity chromatography.
- Example 4 Protein A fixed particles (PA-2) obtained by the same operation as in Example 3 and having an unreacted epoxy group ring-opened were dispersed in pure water so that the concentration of the particles was 10% by mass. To this, 0.48 g of 30% aqueous hydrogen peroxide was added, and the mixture was shaken and stirred at 25 ° C. for 24 hours. Furthermore, it was washed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M aqueous sodium hydroxide solution, and 0.1 M sodium citrate buffer (pH 3.2) to obtain a packing material 4 for affinity chromatography.
- 0.1 M sodium phosphate buffer pH 6.6
- 0.1 M aqueous sodium hydroxide solution 0.1 M sodium citrate buffer
- Example 5 Protein A fixed particles (PA-1) produced in the same manner as in Example 3 were dispersed in 40 mL of 1.0 M 1-thioglycerol / 0.1 M sodium sulfate (pH 8.3) and treated at 25 ° C. for 40 hours. The unreacted epoxy group was opened by shaking and stirring. Further, the obtained protein A-fixed particles in which the unreacted epoxy group was opened were mixed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M sodium hydroxide aqueous solution, 0.1 M sodium citrate buffer (pH 3). . 2) to obtain a packing material 5 for affinity chromatography.
- Example 6 In step (1) of Example 1, it consists of 5.26 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.), 2.63 g of glycerol monomethacrylate (manufactured by NOF Corporation) and 2.63 g of divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.). The same procedure as in steps (1) and (2) of Example 1 was conducted, except that the monomer composition was dissolved in 25.00 g of 2-octanone (Toyo Gosei Co., Ltd.) to prepare a monomer solution. And a porous particle dispersion was obtained.
- protein A was immobilized by the same operation as in step (3) of Example 1 to produce protein A-immobilized particles.
- the produced protein A fixed particles are dispersed in 40 mL of 1.0 M sodium 2-mercaptopropanesulfonate (manufactured by Tokyo Chemical Industry Co., Ltd.) / 0.1 M sodium sulfate (pH 8.3) and shaken at 25 ° C. for 17 hours.
- the unreacted epoxy group was opened by stirring.
- the obtained protein A-fixed particles in which the unreacted epoxy group was opened were mixed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M sodium hydroxide aqueous solution, 0.1 M sodium citrate buffer (pH 3). . 2) to obtain a packing material 6 for affinity chromatography.
- Example 7 In the step (1) of Example 1, a monomer composition consisting of 4.77 g of 4-hydroxybutyl acrylate glycidyl ether (Nippon Kasei Co., Ltd.) and 8.86 g of glycerin dimethacrylate (Shin Nakamura Chemical Co., Ltd.) Except for preparing a monomer solution by dissolving 21.52 g of acetophenone (manufactured by Inoue Fragrance Co., Ltd.) and 7.35 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.), step (1) of Example 1 and The same operation as in (2) was performed to obtain a porous particle dispersion. Thereafter, protein A was immobilized, unreacted epoxy groups were opened, and washed in the same manner as in Example 3 to obtain a packing material 7 for affinity chromatography.
- Example 8 In step (1) of Example 1, 3.43 g of vinyl benzyl glycidyl ether (manufactured by Toray Fine Chemical Co., Ltd.), 4.11 g of glycerol monomethacrylate (manufactured by NOF Corporation) and ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) The monomer composition consisting of 6.17 g was dissolved in a mixed solution of 21.52 g of acetophenone (Inoue Fragrance Co., Ltd.) and 7.35 g of 2-octanone (Toyo Gosei Co., Ltd.) to prepare a monomer solution.
- acetophenone Inoue Fragrance Co., Ltd.
- 2-octanone Toyo Gosei Co., Ltd.
- Example 3 (Comparative Example 1) In Example 3, a filler 9 for affinity chromatography was obtained in the same manner as in Example 3 except that the unreacted epoxy group was not opened after protein A was immobilized.
- Example 2 In the step (1) of Example 1, 1.41 g of 3,4-epoxycyclohexylmethyl methacrylate (manufactured by Daicel), 8.45 g of glycerol monomethacrylate (manufactured by NOF Corporation) and trimethylolpropane trimethacrylate (manufactured by Sartomer) A monomer composition consisting of 4.23 g was dissolved in a mixed solution of 21.50 g of acetophenone (manufactured by Inoue Fragrance Co., Ltd.) and 7.34 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution.
- acetophenone manufactured by Inoue Fragrance Co., Ltd.
- 2-octanone manufactured by Toyo Gosei Co., Ltd.
- Comparative Example 3 In Comparative Example 2, the shaking stirring time after dispersing Protein A fixed particles in 40 mL of 1.0 M 1-thioglycerol (Asahi Chemical Industry Co., Ltd.) / 0.1 M sodium sulfate (pH 8.3) was 4 hours.
- the filler 11 for affinity chromatography was obtained in the same manner as in Comparative Example 2 except that the time was changed to 17 hours.
- step (1) of Example 1 from 1.39 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.), 2.79 g of glycerol monomethacrylate (manufactured by NOF Corporation) and 9.76 g of glycerin dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) Except that the monomer composition was dissolved in a mixed solution of 21.50 g of acetophenone (manufactured by Inoue Fragrance Co., Ltd.) and 7.34 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution.
- glycidyl methacrylate manufactured by Mitsubishi Rayon Co., Ltd.
- glycerol monomethacrylate manufactured by NOF Corporation
- glycerin dimethacrylate manufactured by Shin-Nakamura Chemical Co., Ltd.
- Test Example 1 Quantification of epoxy group content
- the epoxy group content of the porous particles before ligand binding obtained in Examples 1 to 8 and Comparative Examples 1 to 4 was quantified. That is, after adding an excess of hydrochloric acid to porous particles suspended in an aqueous calcium chloride solution to cause addition reaction of hydrochloric acid, the epoxy group is opened, and the remaining hydrochloric acid is neutralized with an excessive amount of aqueous sodium hydroxide solution. The phenolphthalein solution was added, and the remaining sodium hydroxide was quantified by back titrating with hydrochloric acid. The end point of the titration was when the red color of phenolphthalein disappeared. The results are shown in Table 1.
- Test Example 2 Evaluation of residual epoxy group amount (measurement of pH increase)
- the packing material for affinity chromatography obtained in Examples 1 to 8 and Comparative Examples 1 to 4 was packed in a GE Healthcare Tricorn 5/50 column at a packing height of about 5 cm, and the column was manufactured by GE Healthcare.
- a pH 7.5 20 mM sodium phosphate buffer solution containing 2 M NaCl was passed at a flow rate of 0.2 mL / min while monitoring pH by connecting to AKTAprime plus, and when the pH in the column reached equilibrium The pH value was recorded as the initial pH. Further, the column replaced with the buffer solution was removed from AKTAprime plus and allowed to stand in a thermostat at 40 ° C. for 7 days.
- the column was again connected to AKTA prime plus and the buffer was passed at a flow rate of 0.2 mL / min while monitoring the pH, and the pH value when the pH reached the highest was recorded as the load pH. .
- the difference between the initial pH and the load pH was determined as the pH increase range.
- the initial pH and load pH were measured in a constant temperature room at 23 ⁇ 1 ° C. The results are shown in Table 1.
- Test Example 3 Quantification of protein A binding amount
- the amount of protein A, which is a ligand bound to the packing material for affinity chromatography obtained in Examples 1 to 8 and Comparative Examples 1 to 4 was quantified using a bicinchoninic acid (BCA) reagent. Specifically, 1 mg of the filler in terms of solid content was collected in a test tube, and this was quantified with a BCA Protein Assay Kit manufactured by ThermoFisher Scientific. The reaction was performed by inversion mixing at 37 ° C. for 30 minutes. A calibration curve was prepared using the same protein A bound to the carrier. The results are shown in Table 1.
- BCA bicinchoninic acid
- Test Example 4 DBC measurement and storage stability evaluation
- DBC of each of the fillers of Examples and Comparative Examples was measured against a protein (human IgG antibody, HEG-1000 manufactured by Equitech Bio) at a linear flow rate of 300 cm / hr using GE Healthcare's AKTAprime plus.
- a column with a capacity of 4 mL (5 mm ⁇ ⁇ 200 mm length), and a protein with 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) dissolved in 5 mg / mL, and a 10% break at the elution tip.
- DBC was determined from the amount of protein captured during the through and the column packing volume. The results are shown in Table 1.
- Test Example 5 Quality of HCP
- Columns (Tricorn 10/50 column manufactured by GE Healthcare) were filled with each of the fillers of Examples and Comparative Examples at a packing height of about 5 cm to prepare a column.
- Each of the obtained columns was connected to GE Healthcare's AKTA Prime Plus, and 20 mM sodium phosphate buffer (pH 7.5) was passed through at 5 column volumes (5 times the column volume) at a flow rate of 1 mL / min. Equilibrated.
- the CHO cell culture supernatant containing the monoclonal antibody Trastuzumab was passed through the column at a flow rate of 1 mL / min with a loading amount of about 23 mg antibody / mL carrier.
- 20 mM sodium phosphate buffer (pH 7.5), 20 mM sodium phosphate / 1 M sodium chloride buffer (pH 7.5), and 20 mM sodium phosphate buffer (pH 7.5) were each 5 column volumes, flow rate 1 mL / min. Were sequentially passed through the column. Thereafter, 50 mM sodium citrate buffer (pH 3.2) was passed through the column at a flow rate of 1 mL / min to elute the monoclonal antibody captured in the column. An elution fraction of 280> 100 mAu was collected. And the antibody concentration (mg / mL) contained in the collect
- HCP Host Cell Protein
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Abstract
Description
一般に、タンパク精製用のアフィニティークロマトグラフィーに求められる性能としては、(1)標的タンパク質以外の不純物を非特異吸着しないこと、(2)長期間の保管により結合容量が低下せず、保存安定性に優れること、(3)カラム操作する上で充分な機械的強度があること、などが挙げられる。
また、スチレン-ジビニルベンゼンのような芳香族ビニル単量体の共重合体からなる多孔質粒子は、一般に耐アルカリ性に優れるが、親水性が低いために、リガンドの活性が低く、標的物質に対する動的結合容量が低いという欠点を有する。
さらに、メタクリレート系ビニル単量体の重合体からなる多孔質粒子は、高い親水性と機械的強度とを有しているものの、不純物の除去能については更なる改善が望まれていた。
(M-2)ポリビニル単量体に由来する構造単位:全構造単位に対し0.5~80質量部を含む共重合体を含有する固相担体と、
エポキシ基を開環させて得られる開環エポキシ基と、
前記固相担体に結合したリガンドと、を含むアフィニティークロマトグラフィー用担体であって、
前記アフィニティークロマトグラフィー用担体を任意のカラム容器に充填し、カラムを2MのNaClを含むpH7.5の20mMリン酸ナトリウム緩衝液で置換し、40℃で7日間静置したときのカラム内のpH上昇幅が2以下であることを特徴とする、
アフィニティークロマトグラフィー用担体を提供するものである。
したがって、本発明のアフィニティークロマトグラフィー用担体によれば、非特異的に吸着する不純物、例えば宿主細胞タンパク質(HCP=Host Cell Protein)やDNAの除去能を改善することが可能となる。
また、リガンドの活性を高く保つことができるため、本発明のアフィニティークロマトグラフィー用担体を、例えばイムノグロブリンの精製に用いた後で、更に長期間繰り返し使用してもイムノグロブリン等の動的結合容量が低下しにくく、低コストでイムノグロブリン精製が可能となる。
<固相担体の構成>
本発明のアフィニティークロマトグラフィー用担体を構成する固相担体は、
(M-1)エポキシ基含有モノビニル単量体に由来する構造単位:全構造単位に対し20質量部を超えて、99.5質量部以下、および
(M-2)ポリビニル単量体に由来する構造単位:全構造単位に対し0.5~80質量部を含む共重合体を含有し、
且つ、エポキシ基を開環させて得られる開環エポキシ基を含むものである。固相担体の形態は、モノリス、膜、中空繊維、粒子、カセット、チップ等のいずれでもよいが、粒子が好ましい。また、固相担体は、表面積の向上の観点から、多孔質粒子等の多孔質化されたものが好ましい。また、多孔質粒子としては、多孔質ポリマー粒子が好ましい。なお、固相担体は、上記共重合体以外に、天然高分子や合成高分子などを含んでいてもよい。
エポキシ基含有モノビニル単量体は、1分子中に、1個の重合性ビニル基(エチレン性不飽和結合を有する基)と、1個以上のエポキシ基とを有する単量体である。エポキシ基含有ビニル単量体は、固相担体に適切な量のエポキシ基を導入し、適切なリガンド結合量や、開環エポキシ基量を得るために利用可能である。
これらの中でも、エポキシ基含有モノビニル単量体としては、防汚性や保存安定性等の観点から、エポキシ基含有(メタ)アクリル酸エステル類、エポキシ基含有芳香族モノビニル化合物が好ましく、エポキシ基含有(メタ)アクリル酸エステル類がより好ましく、グリシジル(メタ)アクリレート、4-ヒドロキシブチル(メタ)アクリレートグリシジルエーテルがさらに好ましく、グリシジル(メタ)アクリレートが特に好ましい。なお、固相担体を順相懸濁重合により粒子状にする場合には、エポキシ基含有モノビニル単量体としては、開環エポキシ基量のコントロールが容易となることから水不溶性のものが好ましい。ここで水不溶性とは水100mLに対して室温(25℃)で10g以上は溶解しないことをいう。なお、エポキシ基含有モノビニル単量体は市販品を用いてもよく、公知の方法に従い合成して使用してもよい。
エポキシ基含有モノビニル単量体に由来する構造単位の含有量は、防汚性や保存安定性等の観点から、全構造単位に対し、好ましくは25質量部以上、より好ましくは30質量部以上、さらに好ましくは40質量部以上、特に好ましくは50質量部以上であり、また、防汚性や保存安定性等の観点から、全構造単位に対し、好ましくは95質量部以下、より好ましくは90質量部以下、さらに好ましくは85質量部以下、特に好ましくは80質量部以下である。
ポリビニル単量体は、1分子中に、2個以上の重合性ビニル基(エチレン性不飽和結合を有する基)を有するビニル単量体である。以下、当該ポリビニル単量体を、ヒドロキシ基非含有ポリビニル単量体と、ヒドロキシ基含有ポリビニル単量体とに分けて説明する。なお、ポリビニル単量体は、1種を単独でまたは2種以上を組み合わせて使用できる。
また、上記ヒドロキシ基非含有ポリビニル単量体に含まれる重合性ビニル基の個数としては、1分子中に、2~5個が好ましく、2または3個がより好ましい。
多価アルコールの(メタ)アクリル酸エステル類としては、グリセリンジ(メタ)アクリレート、トリメチロールエタンジ(メタ)アクリレート、トリメチロールプロパンジ(メタ)アクリレート、ブタントリオールジ(メタ)アクリレート、ペンタエリスリトールジ(メタ)アクリレート、ペンタエリスリトールトリ(メタ)アクリレート、ジペンタエリスリトールジ(メタ)アクリレート、ジペンタエリスリトールトリ(メタ)アクリレート、ジペンタエリスリトールテトラ(メタ)アクリレート、ジペンタエリスリトールペンタ(メタ)アクリレート、イノシトールジ(メタ)アクリレート、イノシトールトリ(メタ)アクリレート、イノシトールテトラ(メタ)アクリレートなどが挙げられる。
各種糖類の2置換以上の(メタ)アクリル酸エステル類としては、例えば、グルコースジ(メタ)アクリレート、グルコーストリ(メタ)アクリレート、グルコーステトラ(メタ)アクリレート、マンニトールジ(メタ)アクリレート、マンニトールトリ(メタ)アクリレート、マンニトールテトラ(メタ)アクリレート、マンニトールペンタ(メタ)アクリレートなどが挙げられる。
さらに、ヒドロキシ基含有ポリビニル単量体としては、上記例示したものの他に、ジアミノプロパノール、トリスヒドロキシメチルアミノメタン、グルコサミンなどのアミノアルコールと(メタ)アクリル酸との脱水縮合反応物なども挙げることができる。
また、上記ヒドロキシ基含有ポリビニル単量体に含まれる重合性ビニル基の個数としては、1分子中に、2~5個が好ましく、2または3個がより好ましい。
ポリビニル単量体に由来する構造単位の含有量は、防汚性や保存安定性等の観点から、全構造単位に対し、好ましくは5質量部以上、より好ましくは10質量部以上、さらに好ましくは15質量部以上、さらに好ましくは20質量部以上、特に好ましくは25質量部以上であり、また、防汚性や保存安定性等の観点から、全構造単位に対し、好ましくは70質量部以下、より好ましくは65質量部以下、さらに好ましくは60質量部以下、さらに好ましくは50質量部以下、特に好ましくは40質量部以下である。
本発明で用いる固相担体に含まれる共重合体としては、上記構造単位(M-1)及び構造単位(M-2)に加えて、(M-3)エポキシ基を含有しないモノビニル単量体に由来する構造単位:全構造単位に対し40質量部以下を更に含むものが好ましい。
また、エポキシ基を含有しないヒドロキシ基含有モノビニル単量体に含まれるヒドロキシ基の個数としては、1分子中に、1~5個が好ましく、1~3個がより好ましい。
リガンド結合前の固相担体に含まれるエポキシ基は、リガンドを結合するための官能基であり、また、さらに開環後にアフィニティークロマトグラフィー用担体としての親水性向上の元となる官能基である。リガンド結合前の固相担体のエポキシ基含有量は、好ましくは1~6mmol/gであり、さらに好ましくは2~5mmol/gであり、特に好ましくは2.5~4mmol/gである。リガンド結合前の固相担体のエポキシ基含有量が1mmol/g以上であると、リガンド結合後に得られる開環エポキシ基の量が適切な量となり、標的物質以外の不純物の除去能が向上する。リガンド結合前の固相担体のエポキシ基含有量が6mmol/g以下であると、リガンド結合後のエポキシ基の残留量が減少し、アフィニティークロマトグラフィー用担体として保存した場合のリガンドの活性の低下が抑制され、繰り返し使用した際の標的物質の動的結合容量の低下も抑制される。
リガンド結合前の固相担体のエポキシ基含有量は、エポキシ基含有モノビニル単量体の量、重合温度、重合時間、重合液のpH、さらには、重合後の開環処理などによって、調整することが出来る。
固相担体の製造法を、固相担体が多孔質粒子である場合を例に挙げて説明する。
多孔質粒子は、公知のシード重合、懸濁重合などにより製造することができる。シード重合法として、特公昭57-24369号公報記載の二段膨潤重合法が挙げられる。重合に際しては、上記単量体に加えて、重合開始剤、多孔化剤、水系媒体、分散安定剤、界面活性剤、重合調整剤、重合禁止剤、シード粒子などを必要に応じて使用する。
上記多孔化剤の合計使用量は、通常、単量体総量100質量部に対して40~400質量部程度である。
また、亜硝酸ナトリウム等の亜硝酸塩、ヨウ化カリウム等のヨウ化物塩、tert-ブチルピロカテコール、ベンゾキノン、ピクリン酸、ハイドロキノン、塩化銅、塩化第二鉄等の重合禁止剤を用いることもできる。
また、ドデシルメルカプタン等の重合調製剤を用いてもよい。
また、重合時間は通常5分~48時間、好ましくは10分~24時間である。
また、シード粒子および/または多孔化剤の良溶媒を用いて洗浄することが、後述のリガンドの結合が容易となる点から、好ましい。洗浄溶媒としては、アセトン、エタノール、イソプロピルアルコールなどが好適に使用できる。また、必要に応じて、洗浄の前後に、超音波分散機などを用いて、多孔質粒子を分散してもよい。さらに、デカンテーション、ろ過、篩分級などの方法により、小粒子や粗大粒子を除去することが、圧力特性の向上や標的物質などの動的結合容量の向上の点から好ましい。
(開環エポキシ基)
本発明のアフィニティークロマトグラフィー用担体は、エポキシ基を開環させて得られる開環エポキシ基を有する。この開環エポキシ基は、上記共重合体を含有する固相担体にリガンドを結合した後に、該共重合体に含まれるエポキシ基、すなわち、リガンドと結合したエポキシ基以外の残余のエポキシ基を開環させて得ることができる。エポキシ基には上記の共重合体に含まれるものの他、エピクロロヒドリンやジグリシジルエーテル化合物等により、後から導入したエポキシ基も含まれ、本発明のアフィニティークロマトグラフィー用担体の製造においてどの段階で導入されたかは問わない。
そのため、本発明のアフィニティークロマトグラフィー用担体は、アフィニティークロマトグラフィー用担体を任意のカラム容器に充填し、カラムを2MのNaClを含むpH7.5の20mMリン酸ナトリウム緩衝液で置換し、40℃で7日間静置したときのカラム内のpH上昇幅が2以下となるものである。斯かる構成により、不純物の除去能と保存安定性に優れたものとなる。上記pH上昇幅は、好ましくは1.9以下であり、不純物の除去能を更に改善させる場合には、1.7以下がより好ましい。また、pH上昇幅の下限値は特に限定されないが、例えば0.01である。
(pH上昇幅)=(負荷pH)-(初期pH)
上記式において「負荷pH」は、初期pHを測定してから40℃の恒温器内で7日間静置した後、2MのNaClを含むpH7.5の20mMリン酸ナトリウム緩衝液をカラムに再度通液し、pHが最も高くなったときのpHの値を意味する。
R1は、炭素数1~6の2価の有機基を示し、
R2は、水素原子または炭素数1~10の1価の有機基を示し、
Xは、チオ基(>S)、スルフィニル基(>S=O)、スルホニル基(>S(=O)2)、イミノ基(>NH)、またはオキシ基(>O)を示し、
*は、結合位置を示す。]
また、上記2価の有機基は、直鎖状でも分岐鎖状でもよい。また、斯かる2価の有機基としては、2価の炭化水素基、2価の炭化水素基の炭素-炭素原子間にエーテル結合、イミノ基及びエステル結合から選ばれる1種以上を有する基が挙げられ、2価の炭化水素基が好ましい。
上記2価の炭化水素基は、好ましくは2価の脂肪族炭化水素基であり、より好ましくはアルカンジイル基である。
アルカンジイル基の具体例としては、メタン-1,1-ジイル基、エタン-1,1-ジイル基、エタン-1,2-ジイル基、プロパン-1,1-ジイル基、プロパン-1,2-ジイル基、プロパン-1,3-ジイル基、プロパン-2,2-ジイル基、ブタン-1,4-ジイル基、ペンタン-1,5-ジイル基、ヘキサン-1,6-ジイル基等が挙げられる。
R3は、炭素数1~10の2価または3価の有機基を示し、
nは、1または2を示し、
**は、式(1)中のXとの結合位置を示す。]
R4は、炭素数1~10の2価または3価の有機基を示し、
Yは、酸性基またはアミノ基を示し、
mは、1または2を示し、
**は、式(1)中のXとの結合位置を示す。]
式(3)中、mは、1または2を示すが、1が好ましい。
また、メルカプトエタノール、チオグリセロール、メルカプト酢酸やその塩、メルカプトコハク酸やその塩、メルカプトエタンスルホン酸やその塩、メルカプトプロパンスルホン酸やその塩などの親水性基を有するメルカプト化合物;モノエタノールアミン、グルコサミンなどの親水性基を有するアミン化合物;ジエチレングリコール、グリセリンなどの多価アルコール類をエポキシ基を開環させるブロッキング剤として使用してもよい。なお、ここでいう親水性基としては、ヒドロキシ基;アミノ基;カルボキシ基、リン酸基、スルホ基、これらの塩等の酸性基などが挙げられる。塩としては、ナトリウム塩、カリウム塩等のアルカリ金属塩;マグネシウム塩、カルシウム塩等のアルカリ土類金属塩;アンモニウム塩;有機アンモニウム塩等が挙げられる。
また、必要に応じて塩基性触媒存在下でブロッキングを行ってもよい。斯かる塩基性触媒としては、トリエチルアミン、N,N-ジメチル-4-アミノピリジン、ジイソプロピルエチルアミンなどが挙げられ、1種を単独でまたは2種以上を組み合わせて使用できる。
また、メルカプト化合物をブロッキング剤として使用した場合、酸化剤を用いてチオ基をスルフィニル基に酸化し、さらに親水性を向上させることができる。
また、酸化剤の合計使用量は、チオ基1モルに対し、通常0.1~10モル当量程度であるが、好ましくは0.5~3モル当量である。
上記溶媒の合計使用量は、原料となる固相担体に対し、通常1~50質量倍程度であるが、好ましくは5~15質量倍である。
本発明のアフィニティークロマトグラフィー用担体は、固相担体に結合したリガンドを含むものである。
リガンドは、標的物質に対して適度なアフィニティーを有するものであれば、その種類は特に限定されないが、例えば、プロテインA、プロテインG、プロテインL、Fc結合タンパク、アビジン等のタンパク質;インシュリン等のペプチド;モノクローナル抗体等の抗体;酵素;ホルモン;DNA;RNA;ヘパリン、ルイスX、ガングリオシド等の糖質;イミノジ酢酸、合成色素、2-アミノフェニル硼素酸、4-アミノベンズアミジン、グルタチオン、ビオチンやその誘導体のような低分子化合物を用いることができる。上記に例示したリガンドはその全体を用いてもよいが、リコンビナント、酵素処理等によって得られるそのフラグメントを用いてもよい。また、人工的に合成されたペプチドやペプチド誘導体であってもよい。
イムノグロブリン結合性タンパク質としては、プロテインA、プロテインG、プロテインL、Fc結合タンパクおよびそれらの機能性変異体よりなる群から選ばれる少なくとも1種以上が好ましい。中でも、好ましくはプロテインA、プロテインG、それらの機能性変異体であり、より好ましくはプロテインA、その機能性変異体である。
本発明のアフィニティークロマトグラフィー用担体を構成する固相担体が、多孔質粒子である場合、上記多孔質粒子の粒径は、通常、35~100μmであり、好ましくは40~85μmである。粒径が、35μm以上であると、圧力特性に優れる。100μm以下であると、動的結合容量が高い充填剤が得られる。
多孔質粒子の粒径は、重合する際の条件で調整することができる。なお、本発明における「粒径」とは、レーザ回折散乱式粒度分布測定装置(ベックマン・コールター社製LS13 320)により得られる体積平均粒子径を意味する。
本発明のアフィニティークロマトグラフィーカラムは、本発明のアフィニティークロマトグラフィー用担体がカラム容器に充填されたものである。
本発明の標的物質の精製方法は、本発明のアフィニティークロマトグラフィー用担体を用いることを特徴とするものである。
精製は、本発明のアフィニティークロマトグラフィー用担体を用いる以外は常法と同様であるが、例えば、標的物質を含む組成物を準備する準備工程と、本発明のクロマトグラフィーカラムに前記組成物を通液する通液工程と、該通液工程により担体に吸着された標的物質を溶出させる溶出工程を含む方法が挙げられる。
本発明の精製方法は、タンパク質の精製に適し、イムノグロブリンの精製に特に適する。
(1)360gの純水にポリビニルアルコール(クラレ社製 PVA-217)3.58gを添加し、加熱撹拌してポリビニルアルコールを溶解させ、冷却した後、ドデシル硫酸ナトリウム(和光純薬工業製)0.36g、硫酸ナトリウム(和光純薬工業製)0.36g、亜硝酸ナトリウム(和光純薬工業製)0.18gを添加し、撹拌して水溶液Sを調製した。
一方、グリシジルメタクリレート(三菱レーヨン社製)12.00g及びジビニルベンゼン(新日鐵化学社製)1.33gからなる単量体組成物を、ジイソブチルケトン(三井化学社製)24.43gに溶解させ、単量体溶液を調製した。
次いで、前記水溶液Sを、500mLセパラブルフラスコ内に全量投入し、温度計、撹拌翼及び冷却管を装着して、温水バスにセットし、窒素雰囲気下で撹拌を開始した。セパラブルフラスコ内に前記単量体溶液を全量投入して、温水バスにより加温し内温が85℃に到達したところで2,2'-アゾイソブチロニトリル(和光純薬工業社製)0.53gを添加し、86℃に温度を維持した。
(2)その後、86℃に温度を維持し、3時間撹拌を行った。次いで、反応液を冷却した後、斯かる反応液をろ過し、純水とエタノールで洗浄した。洗浄した粒子を純水に分散させてデカンテーションを3回行い、小粒子を除いた。次いで、粒子の濃度が10質量%となるように粒子を純水に分散させ、多孔質粒子分散液を得た。
(3)改変プロテインA(Repligen製 rSPA)0.15gを、1.2M 硫酸ナトリウム/0.1M リン酸ナトリウムバッファー(pH6.6)40mLに分散させプロテインA分散液を得、このプロテインA分散液に実施例1の工程(2)で得られた多孔質粒子分散液(粒子乾燥質量換算で1g相当)を添加した。この分散液を25℃で5時間振とう撹拌し、プロテインAを粒子に固定した。
(4)生成したプロテインA固定粒子を、0.1M リン酸ナトリウムバッファー(pH6.6)で洗浄した後、1.0M 硫酸水溶液(和光純薬工業社製)40mLに分散させ、40℃で4時間振とう撹拌することで、未反応のエポキシ基を開環させた。
その後、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤1を得た。
実施例1の工程(1)において、グリシジルメタクリレート(三菱レーヨン社製)9.20g、1-エチル-4-ビニルベンゼン(ChemSampCo社製)0.58g及びジビニルベンゼン(新日鐵化学社製)1.73gからなる単量体組成物を、ジイソブチルケトン(三井化学社製)19.59g及びアニソール(和光純薬工業社製)6.05gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例1の工程(3)と同様の操作でプロテインAを固定し、プロテインA固定粒子を生成させた。
次いで、生成したプロテインA固定粒子を、1.0M 2-メルカプトエタノール(和光純薬工業社製)/0.1M 硫酸ナトリウム(pH8.3)40mLに分散させ、25℃で17時間振とう撹拌することで、未反応のエポキシ基を開環させた。さらに、得られた未反応のエポキシ基が開環したプロテインA固定粒子を、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤2を得た。
実施例1の工程(1)において、グリシジルメタクリレート(三菱レーヨン社製)8.18g、N-(2-ヒドロキシエチル)アクリルアミド(東京化成工業社製)1.36g及びトリメチロールプロパントリメタクリレート(サートマー社製)4.09gからなる単量体組成物を、4-メチル-2-ペンタノール(和光純薬工業製)21.97g及び炭酸ジエチル(和光純薬工業製)2.95gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例1の工程(3)と同様の操作でプロテインAを固定し、プロテインA固定粒子を生成させた。
次いで、生成したプロテインA固定粒子(PA-1)を、1.0M 1-チオグリセロール(旭化学工業社製)/0.1M 硫酸ナトリウム(pH8.3)40mLに分散させ、25℃で17時間振とう撹拌することで、未反応のエポキシ基を開環させた。さらに、得られた未反応のエポキシ基が開環したプロテインA固定粒子(PA-2)を、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤3を得た。
実施例3と同様の操作で得た、未反応のエポキシ基が開環したプロテインA固定粒子(PA-2)を、粒子の濃度が10質量%となるように純水に分散させた。これに、30%過酸化水素水0.48gを添加し、25℃で24時間振とう撹拌した。さらに、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤4を得た。
実施例3と同様の操作で生成させたプロテインA固定粒子(PA-1)を、1.0M 1-チオグリセロール/0.1M 硫酸ナトリウム(pH8.3)40mLに分散させ、25℃で40時間振とう撹拌することで、未反応のエポキシ基を開環させた。さらに、得られた未反応のエポキシ基が開環したプロテインA固定粒子を、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤5を得た。
実施例1の工程(1)において、グリシジルメタクリレート(三菱レーヨン社製)5.26g、グリセロールモノメタクリレート(日油社製)2.63g及びジビニルベンゼン(新日鐵化学社製)2.63gからなる単量体組成物を、2-オクタノン(東洋合成社製)25.00gに溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例1の工程(3)と同様の操作でプロテインAを固定し、プロテインA固定粒子を生成させた。
次いで、生成したプロテインA固定粒子を、1.0M 2-メルカプトプロパンスルホン酸ナトリウム(東京化成工業社製)/0.1M 硫酸ナトリウム(pH8.3)40mLに分散させ、25℃で17時間振とう撹拌することで、未反応のエポキシ基を開環させた。さらに、得られた未反応のエポキシ基が開環したプロテインA固定粒子を、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤6を得た。
実施例1の工程(1)において、4-ヒドロキシブチルアクリレートグリシジルエーテル(日本化成社製)4.77g及びグリセリンジメタクリレート(新中村化学工業社製)8.86gからなる単量体組成物を、アセトフェノン(井上香料製造所社製)21.52g及び2-オクタノン(東洋合成社製)7.35gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例3と同様の操作でプロテインAの固定、未反応のエポキシ基の開環、及び洗浄を実施し、アフィニティークロマトグラフィー用充填剤7を得た。
実施例1の工程(1)において、ビニルベンジルグリシジルエーテル(東レ・ファインケミカル社製)3.43g、グリセロールモノメタクリレート(日油社製)4.11g及びエチレングリコールジメタクリレート(新中村化学工業社製)6.17gからなる単量体組成物を、アセトフェノン(井上香料製造所社製)21.52g及び2-オクタノン(東洋合成社製)7.35gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例3と同様の操作でプロテインAの固定、未反応のエポキシ基の開環、及び洗浄を実施し、アフィニティークロマトグラフィー用充填剤8を得た。
実施例3において、プロテインA固定後に未反応のエポキシ基の開環を行わないこと以外は、実施例3と同様の操作でアフィニティークロマトグラフィー用充填剤9を得た。
実施例1の工程(1)において、3,4-エポキシシクロヘキシルメチルメタクリレート(ダイセル社製)1.41g、グリセロールモノメタクリレート(日油社製)8.45g及びトリメチロールプロパントリメタクリレート(サートマー社製)4.23gからなる単量体組成物を、アセトフェノン(井上香料製造所社製)21.50g及び2-オクタノン(東洋合成社製)7.34gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例1の工程(3)と同様の操作でプロテインAを固定し、プロテインA固定粒子を生成させた。
次いで、生成したプロテインA固定粒子を、1.0M 1-チオグリセロール(旭化学工業社製)/0.1M 硫酸ナトリウム(pH8.3)40mLに分散させ、25℃で4時間振とう撹拌することで、未反応のエポキシ基を開環させた。さらに、0.1M リン酸ナトリウムバッファー(pH6.6)、0.1M 水酸化ナトリウム水溶液、0.1Mクエン酸ナトリウムバッファー(pH3.2)で洗浄し、アフィニティークロマトグラフィー用充填剤10を得た。
比較例2において、1.0M 1-チオグリセロール(旭化学工業社製)/0.1M 硫酸ナトリウム(pH8.3)40mLにプロテインA固定粒子を分散させた後の振とう撹拌時間を、4時間から17時間に変更した以外は、比較例2と同様の操作でアフィニティークロマトグラフィー用充填剤11を得た。
実施例1の工程(1)において、グリシジルメタクリレート(三菱レーヨン社製)1.39g、グリセロールモノメタクリレート(日油社製)2.79g及びグリセリンジメタクリレート(新中村化学工業社製)9.76gからなる単量体組成物を、アセトフェノン(井上香料製造所社製)21.50g及び2-オクタノン(東洋合成社製)7.34gの混液に溶解させ、単量体溶液を調製した以外は、実施例1の工程(1)及び(2)と同様の操作を行い、多孔質粒子分散液を得た。
その後、実施例3と同様の操作でプロテインAの固定、未反応のエポキシ基の開環、及び洗浄を実施し、アフィニティークロマトグラフィー用充填剤12を得た。
実施例1~8および比較例1~4で得られたリガンド結合前の多孔質粒子のエポキシ基含有量を定量した。
すなわち、塩化カルシウム水溶液に懸濁した多孔質粒子に塩酸を過剰に添加して塩酸を付加反応させることによりエポキシ基を開環し、残余の塩酸を過剰量の水酸化ナトリウム水溶液で中和した後、フェノールフタレイン溶液を添加し、残余の水酸化ナトリウムを塩酸で逆滴定することにより定量した。なお、滴定の終点はフェノールフタレインの赤色が消えた時点とした。結果を表1に示す。
実施例1~8および比較例1~4で得られたアフィニティークロマトグラフィー用充填剤をGEヘルスケア社製Tricorn 5/50カラムに充填高さ約5cmで充填し、前記カラムをGEヘルスケア社製AKTAprime plusに接続してpHをモニタリングしながら2MのNaClを含むpH7.5の20mMリン酸ナトリウム緩衝液を流速0.2mL/分にて通液し、カラム内のpHが平衡に達したときのpHの値を初期pHとして記録した。さらに、前記緩衝液に置換した前記カラムをAKTAprime plusから取り外し、40℃の恒温器内で7日間静置した。
その後、前記カラムを再びAKTAprime plusに接続してpHをモニタリングしながら前記緩衝液を流速0.2mL/分にて通液し、pHが最も高くなったときのpHの値を負荷pHとして記録した。初期pHと負荷pHとの差をpH上昇幅として求めた。なお、初期pH及び負荷pHの測定は23±1℃の恒温室内で実施した。結果を表1に示す。
実施例1~8および比較例1~4で得られたアフィニティークロマトグラフィー用充填剤に結合したリガンドであるプロテインAの結合量を、ビシンコニン酸(BCA)試薬を用いて定量した。具体的には、固形分換算で1mgの充填剤をテストチューブに採取し、これをThermoFisher Scientific社のBCA Protein Assay Kitで定量した。反応は、37℃で30分間、転倒混和することによって行った。検量線は、担体に結合させたプロテインAと同一のものを用いて作成した。結果を表1に示す。
GEヘルスケア社製AKTAprime plusを用いて、線流速300cm/hrにおけるタンパク質(ヒトIgG抗体、Equitech Bio社製 HGG-1000)に対する実施例および比較例の各充填剤のDBCを測定した。カラムは容量4mL(5mmφ×200mm長)のものを、タンパク質は20mMリン酸ナトリウム/150mM塩化ナトリウム水溶液(pH7.5)で5mg/mLにタンパク質を溶解したものをそれぞれ使用し、溶出先端10%ブレークスルーのときのタンパク質捕捉量とカラム充填体積からDBCを求めた。結果を表1に示す。
カラム容器(GEヘルスケア社製Tricorn10/50 column)に、実施例及び比較例の各充填剤を、充填高さ約5cmで充填してカラムを作製した。得られたカラムをGEヘルスケア社製AKTA Prime Plusにそれぞれ接続し、20mMリン酸ナトリウムバッファー(pH7.5)を5カラム容量(カラム容積の5倍)、流速1mL/分にて通液し、平衡化させた。
次いで、モノクローナル抗体Trastuzumabを含有するCHO細胞培養上清を、約23mg抗体/mL担体の負荷量で、流速1mL/分にてカラムに通液した。
次いで、20mMリン酸ナトリウムバッファー(pH7.5)、20mMリン酸ナトリウム/1M塩化ナトリウムバッファー(pH7.5)、及び20mMリン酸ナトリウムバッファー(pH7.5)を、それぞれ5カラム容量、流速1mL/分にてカラムに順次通液した。
その後、50mMクエン酸ナトリウムバッファー(pH3.2)を、流速1mL/分にてカラムに通液し、カラム内に捕捉されていたモノクローナル抗体を溶出させ、Abs.280>100mAuの溶出フラクションを回収した。
そして、分光光度計を用い、回収したフラクション中に含有される抗体濃度(mg/mL)を測定した。また、Cygnus Technologies社製 CHO HCP ELISA kit,3Gを用い、回収したフラクション中に含有されるHost Cell Protein(HCP)の濃度(ng/mL)を測定した。さらにHCPの濃度を抗体濃度で除することにより、単位抗体量当たりのHCP量を算出した。結果を表1に示す。
(M-1) GMA:グリシジルメタクリレート、HBAGE:4-ヒドロキシブチルアクリレートグリシジルエーテル、VBGE:(4-ビニルベンジル)グリシジルエーテル、METHB:3,4-エポキシシクロヘキシルメチルメタクリレート
(M-2) DVB:ジビニルベンゼン、TMP:トリメチロールプロパントリメタクリレート、GLDM:グリセリンジメタクリレート、EDMA:エチレングリコールジメタクリレート
(M-3) EVB:1-エチル-4-ビニルベンゼン、HEAA:N-(2-ヒドロキシエチル)メタクリルアミド、GLM:グリセロールモノメタクリレート
(開環に使用した化合物) ME:2-メルカプトエタノール、TG:1-チオグリセロール、MPSA:2-メルカプトプロパンスルホン酸ナトリウム
Claims (12)
- (M-1)エポキシ基含有モノビニル単量体に由来する構造単位:全構造単位に対し20質量部を超えて、99.5質量部以下、および
(M-2)ポリビニル単量体に由来する構造単位:全構造単位に対し0.5~80質量部を含む共重合体を含有する固相担体と、
エポキシ基を開環させて得られる開環エポキシ基と、
前記固相担体に結合したリガンドと、を含むアフィニティークロマトグラフィー用担体であって、
前記アフィニティークロマトグラフィー用担体を任意のカラム容器に充填し、カラムを2MのNaClを含むpH7.5の20mMリン酸ナトリウム緩衝液で置換し、40℃で7日間静置したときのカラム内のpH上昇幅が2以下であることを特徴とする、
アフィニティークロマトグラフィー用担体。 - 前記共重合体が、(M-3)エポキシ基を含有しないモノビニル単量体に由来する構造単位:全構造単位に対し40質量部以下を更に含む、請求項1に記載のアフィニティークロマトグラフィー用担体。
- Xが、チオ基(>S)、スルフィニル基(>S=O)、またはオキシ基(>O)である、請求項3または4に記載のアフィニティークロマトグラフィー用担体。
- 前記リガンドが、イムノグロブリン結合性タンパク質である、請求項1~5のいずれか1項に記載のアフィニティークロマトグラフィー用担体。
- 前記イムノグロブリン結合性タンパク質が、プロテインA、プロテインG、プロテインL、Fc結合タンパクおよびそれらの機能性変異体よりなる群から選ばれる少なくとも1種以上である、請求項6に記載のアフィニティークロマトグラフィー用担体。
- 前記固相担体が多孔質粒子である、請求項1~7のいずれか1項に記載のアフィニティークロマトグラフィー用担体。
- 前記多孔質粒子の粒径が35~100μmである、請求項8に記載のアフィニティークロマトグラフィー用担体。
- 請求項1~9のいずれか1項に記載のアフィニティークロマトグラフィー用担体がカラム容器に充填された、アフィニティークロマトグラフィーカラム。
- 請求項1~9のいずれか1項に記載のアフィニティークロマトグラフィー用担体を用いることを特徴とする、標的物質の精製方法。
- 前記標的物質が、標的タンパク質である、請求項11に記載の精製方法。
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EP15812576.5A EP3162809B1 (en) | 2014-06-27 | 2015-06-25 | Carrier for affinity chromatography |
US15/322,024 US20170145051A1 (en) | 2014-06-27 | 2015-06-25 | Carrier for affinity chromatography |
CN201580034162.XA CN106459141B (zh) | 2014-06-27 | 2015-06-25 | 亲和色谱用载体 |
KR1020167035941A KR102410991B1 (ko) | 2014-06-27 | 2015-06-25 | 친화성 크로마토그래피용 담체 |
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JP2016044138A (ja) * | 2014-08-22 | 2016-04-04 | Jsr株式会社 | 担体、担体の製造方法、及び標的物の精製方法 |
WO2017146225A1 (ja) * | 2016-02-26 | 2017-08-31 | Jsr株式会社 | アフィニティクロマトグラフィー用多孔質担体、リガンド結合多孔質担体、標的物の精製方法、及び抗体 |
WO2018155241A1 (ja) * | 2017-02-27 | 2018-08-30 | 昭和電工株式会社 | サイズ排除クロマトグラフィー用の充填剤およびその製造方法 |
WO2020004583A1 (ja) | 2018-06-29 | 2020-01-02 | 三菱ケミカル株式会社 | ポリペプチドの分離方法、ポリペプチドの製造方法及びポリペプチドの精製装置 |
WO2020045477A1 (ja) * | 2018-08-29 | 2020-03-05 | Jsr株式会社 | ポリペプチドの精製方法 |
JPWO2019004440A1 (ja) * | 2017-06-30 | 2020-04-30 | 昭和電工株式会社 | 液体クロマトグラフィー用充填剤及び液体クロマトグラフィー用カラム |
JPWO2019208672A1 (ja) * | 2018-04-27 | 2021-05-27 | キヤノン株式会社 | 粒子、粒子の製造方法、アフィニティー粒子、及び、これを含む試薬及びキット、並びに標的物質の検出方法 |
WO2023058409A1 (ja) | 2021-10-05 | 2023-04-13 | Jsr株式会社 | クロマトグラフィー担体をカラムに充填する方法、スラリーの保存方法、及びスラリー |
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WO2015080174A1 (ja) * | 2013-11-27 | 2015-06-04 | Jsr株式会社 | 固相担体、固相担体の製造方法、アフィニティ精製用担体、アフィニティクロマトグラフィー用充填剤の製造方法、アフィニティクロマトグラフィー用充填剤、クロマトグラフィーカラムおよび精製方法 |
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- 2015-06-25 WO PCT/JP2015/068403 patent/WO2015199196A1/ja active Application Filing
- 2015-06-25 JP JP2016529662A patent/JPWO2015199196A1/ja active Pending
- 2015-06-25 KR KR1020167035941A patent/KR102410991B1/ko active IP Right Grant
- 2015-06-25 CN CN201580034162.XA patent/CN106459141B/zh active Active
- 2015-06-26 TW TW104120750A patent/TWI682936B/zh active
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JP2016044138A (ja) * | 2014-08-22 | 2016-04-04 | Jsr株式会社 | 担体、担体の製造方法、及び標的物の精製方法 |
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US11285404B2 (en) | 2017-02-27 | 2022-03-29 | Showa Denko K.K. | Packing material for size exclusion chromatography and method for producing the same |
JPWO2019004440A1 (ja) * | 2017-06-30 | 2020-04-30 | 昭和電工株式会社 | 液体クロマトグラフィー用充填剤及び液体クロマトグラフィー用カラム |
JP7094958B2 (ja) | 2017-06-30 | 2022-07-04 | 昭和電工株式会社 | 液体クロマトグラフィー用充填剤及び液体クロマトグラフィー用カラム |
JPWO2019208672A1 (ja) * | 2018-04-27 | 2021-05-27 | キヤノン株式会社 | 粒子、粒子の製造方法、アフィニティー粒子、及び、これを含む試薬及びキット、並びに標的物質の検出方法 |
JP7305623B2 (ja) | 2018-04-27 | 2023-07-10 | キヤノン株式会社 | 粒子、粒子の製造方法、アフィニティー粒子、及び、これを含む試薬及びキット、並びに標的物質の検出方法 |
WO2020004583A1 (ja) | 2018-06-29 | 2020-01-02 | 三菱ケミカル株式会社 | ポリペプチドの分離方法、ポリペプチドの製造方法及びポリペプチドの精製装置 |
WO2020045477A1 (ja) * | 2018-08-29 | 2020-03-05 | Jsr株式会社 | ポリペプチドの精製方法 |
WO2023058409A1 (ja) | 2021-10-05 | 2023-04-13 | Jsr株式会社 | クロマトグラフィー担体をカラムに充填する方法、スラリーの保存方法、及びスラリー |
Also Published As
Publication number | Publication date |
---|---|
CN106459141A (zh) | 2017-02-22 |
KR20170020372A (ko) | 2017-02-22 |
TWI682936B (zh) | 2020-01-21 |
US20170145051A1 (en) | 2017-05-25 |
EP3162809B1 (en) | 2021-08-04 |
CN106459141B (zh) | 2019-09-13 |
JPWO2015199196A1 (ja) | 2017-04-20 |
EP3162809A1 (en) | 2017-05-03 |
EP3162809A4 (en) | 2018-01-24 |
KR102410991B1 (ko) | 2022-06-17 |
TW201604205A (zh) | 2016-02-01 |
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