WO2017146225A1 - Support poreux pour chromatographie d'affinité, support poreux conjugué à un ligand, procédé de purification de cible, et anticorps - Google Patents

Support poreux pour chromatographie d'affinité, support poreux conjugué à un ligand, procédé de purification de cible, et anticorps Download PDF

Info

Publication number
WO2017146225A1
WO2017146225A1 PCT/JP2017/007172 JP2017007172W WO2017146225A1 WO 2017146225 A1 WO2017146225 A1 WO 2017146225A1 JP 2017007172 W JP2017007172 W JP 2017007172W WO 2017146225 A1 WO2017146225 A1 WO 2017146225A1
Authority
WO
WIPO (PCT)
Prior art keywords
monomer
porous carrier
meth
component
acrylate
Prior art date
Application number
PCT/JP2017/007172
Other languages
English (en)
Japanese (ja)
Inventor
松田　隆志
幸子 津田
智哉 則信
政暁 花村
Original Assignee
Jsr株式会社
Ｊｓｒライフサイエンス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jsr株式会社, Ｊｓｒライフサイエンス株式会社 filed Critical Jsr株式会社
Publication of WO2017146225A1 publication Critical patent/WO2017146225A1/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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 porous carrier for affinity chromatography, a ligand-bound porous carrier, a target purification method, and an antibody.
  • Affinity chromatography plays an important role in the research, development and production of proteins including monoclonal antibodies. 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. Economical purification can be achieved at a high yield with a high yield. Monoclonal antibodies, which are the main components of antibody drugs, are expressed in the culture medium as recombinant proteins mainly using cultured mammalian cells, etc., and are formulated after being purified to high purity by several stages of chromatography and membrane processes. It becomes. With the recent increase in the production amount of antibody drugs, there is a demand for a porous carrier that can withstand treatment at a high flow rate.
  • the problem to be solved by the present invention is to provide a porous carrier for affinity chromatography excellent in the balance between hardness and toughness.
  • Another problem to be solved by the present invention is to provide a porous support for affinity chromatography, a ligand-bound porous support, and a purification method, which have an excellent balance between hardness and toughness and can provide excellent aggregate removal efficiency. It is to provide.
  • Component A including a polymer having a monomer unit derived from a polyvinyl monomer, and (Component B) a monomer unit having an alkyl group having 1 to 8 carbon atoms in the side chain
  • the content ratio [(A) :( B)] with the component B is 95: 5 to 50:50 in terms of mass ratio, and the content of the component A is 0.8% relative to all monomer units in the polymer.
  • a porous support for affinity chromatography hereinafter, also referred to as “the porous support for affinity chromatography of the present invention”
  • the porous support for affinity chromatography of the present invention characterized in that it is 5 to 40% by mass.
  • Component C is a monomer unit derived from glycidyl (meth) acrylate, a monomer unit derived from glycerol mono (meth) acrylate, a monomer unit derived from 4-hydroxybutyl (meth) acrylate glycidyl ether, 3,4- The monomer unit derived from epoxycyclohexylmethyl (meth) acrylate, the monomer unit derived from vinylbenzyl glycidyl ether, and one or more selected from monomer units derived from styrene, according to [9] or [10] above Porous carrier.
  • the porous carrier according to any one of the above [1] to [11], which is in the form of particles, monolith, plate, fiber, or film.
  • a ligand-bound porous carrier obtained by binding an affinity ligand to the porous carrier according to any one of [1] to [12] (hereinafter also referred to as “the ligand-bound porous carrier of the present invention”).
  • [14] A target purification method using the ligand-bound porous carrier according to [13].
  • [15] A step of contacting the ligand-bound porous carrier according to [13] above with a target that can be captured by the affinity ligand, a dissociation solution that dissociates the affinity ligand and the target, and A method for purifying a target product, comprising: a step B in which the carrier that has captured the target product in step A is contacted to dissociate the target product.
  • Step B is a stepwise method in which two or more salt concentrations at which the ionic strength increases stepwise under conditions where the target is dissociated from the affinity ligand, or a graph in which the salt concentration is gradually increased.
  • the purification method according to [15] above which is a step of dissociating by a gent method.
  • a porous carrier for affinity chromatography having an excellent balance between hardness and toughness could be provided. Further, according to the present invention, it was possible to provide a porous carrier for affinity chromatography, a ligand-bound porous carrier, and a purification method that are excellent in the balance between hardness and toughness, and that can provide further excellent aggregate removal efficiency.
  • the porous carrier for affinity chromatography of the present invention comprises a polymer having (Component A) a monomer unit derived from a polyvinyl monomer and (Component B) a monomer unit having an alkyl group having 1 to 8 carbon atoms in the side chain.
  • the content ratio [(A) :( B)] of component A and component B in the polymer is 95: 5 to 50:50 by mass ratio, and the content of component A is It is characterized by being 0.5 to 40% by mass with respect to all monomer units.
  • porous carrier for affinity chromatography and the ligand-bound porous carrier of the present invention will be described first, and then the method for purifying the target substance of the present invention (hereinafter also referred to as the purification method of the present invention).
  • the description of a to b and the like representing a numerical range is synonymous with a to b and below, and a and b are included in the numerical range.
  • the polymer constituting the porous carrier for affinity chromatography of the present invention is preferably insoluble in water.
  • a synthetic polymer is preferred.
  • aromatic monomers such as styrenes; vinyl alcohols; (meth) acrylates; (meth) acrylamides; ethylenes; These are collectively referred to as monomer group ⁇ ).
  • the polymer may contain one or more of the monomer units derived from these monomers, and the porous carrier for affinity chromatography of the present invention comprises such a polymer. 1 type may be included or 2 or more types may be included.
  • the porous carrier for affinity chromatography preferably contains the above polymer as a main component, and the content of the polymer is preferably 70 to 100% by mass, more preferably based on the total amount of the porous carrier for affinity chromatography. 90 to 100% by mass.
  • 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 may be any monomer having two or more polymerizable vinyl groups in one molecule among the monomers corresponding to the monomer group ⁇ , and includes aromatic polyfunctional monomers, polyfunctional (meth) acrylates, and the like.
  • One or more monomers selected from polyfunctional (meth) acrylamides are preferred, and one or more monomers selected from aromatic polyfunctional monomers and polyfunctional (meth) acrylates are more preferred.
  • polyvinyl monomer will be described by dividing it 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.
  • hydroxy group-free polyvinyl monomer examples include ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, and propylene glycol di (meth).
  • polyfunctional (meth) acrylates and aromatic polyfunctional monomers are preferable.
  • polyfunctional (meth) acrylates (meth) acrylic acid esters of polyhydric alcohols 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 dehydration condensation reaction products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth) acrylic acid in addition to those exemplified above.
  • the number of polymerizable vinyl groups contained in the hydroxy group-containing polyvinyl monomer is preferably 2 to 5, more preferably 2 or 3, and particularly preferably 2 in one molecule.
  • polyvinyl monomer good porosity and mechanical strength can be obtained with a relatively small amount of use, and there is little limitation on the amount of epoxy group-containing monovinyl monomer used, so that no hydroxy group is contained.
  • Polyvinyl monomer is preferable, trimethylolpropane tri (meth) acrylate, ethylene glycol di (meth) acrylate, and divinylbenzene are more preferable, and trimethylolpropane tri (meth) acrylate and divinylbenzene are particularly preferable.
  • the content of component A is 0.5 to 40% by mass with respect to all monomer units in the polymer.
  • the content of component A is preferably 1% by mass or more, more preferably 5% by mass or more, and particularly preferably 10% by mass or more with respect to all monomer units in the polymer. Is preferably 35% by mass or less, more preferably 30% by mass or less, still more preferably 25% by mass or less, and particularly preferably 20% by mass or less.
  • the content of each monomer unit in the polymer may be measured by NMR or the like.
  • Component B Monomer unit having an alkyl group having 1 to 8 carbon atoms in the side chain
  • Component B is derived from a monomer having an alkyl group having 1 to 8 carbon atoms in the side chain when polymerized. When the number of carbon atoms of the alkyl group of the monomer exceeds 8, nonspecific adsorption increases.
  • the monomer is preferably a nonionic monomer from the viewpoint of preventing nonspecific adsorption of impurities during purification. Moreover, a monovinyl monomer is preferable.
  • Examples of the monomer for deriving component B include (meth) acrylates having an alkyl group having 1 to 8 carbon atoms, (meth) acrylamides having an alkyl group having 1 to 8 carbon atoms, and alkyl having 1 to 8 carbon atoms.
  • An aromatic monomer having a group can be mentioned, and one kind can be used alone, or two or more kinds can be used in combination.
  • (meth) acrylates having an alkyl group having 1 to 8 carbon atoms and aromatic monomers having an alkyl group having 1 to 8 carbon atoms are preferable.
  • the alkyl group having 1 to 8 carbon atoms is preferably an alkyl group having 2 to 8 carbon atoms.
  • the alkyl group may be linear, branched or cyclic.
  • Examples include a cyclohexyl group, a heptyl group, a cycloheptyl group, an octyl group, a cyclooctyl group, and the like.
  • the alkyl group preferably has no substituent such as a hydrophilic group.
  • the alkyl group is preferably bonded to the main chain of the polymer via a divalent linking group.
  • the divalent linking group include a divalent aromatic hydrocarbon group (such as a phenylene group), an ester bond, and an amide bond.
  • an alkyl (meth) acrylate having an alkyl group having 1 to 8 carbon atoms is preferable.
  • methyl (meth) acrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, 4-tert-butyl (meth) acrylate, isobutyl (meth) acrylate, cyclohexyl (meth) acrylate, n-octyl ( (Meth) acrylate is preferred.
  • the aromatic monomer having an alkyl group having 1 to 8 carbon atoms is preferably an aromatic vinyl compound having an alkyl group having 1 to 8 carbon atoms, and a styrene monomer having an alkyl group having 1 to 8 carbon atoms. Is more preferable.
  • the monomer unit represented by following formula (5) is preferable as a monomer unit derived from the said aromatic monomer.
  • R 8 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms
  • R 9 represents an alkyl group having 1 to 8 carbon atoms
  • t represents an integer of 1 to 5.
  • the number of carbon atoms of the alkyl group represented by R 8 is preferably 1 or 2.
  • the alkyl group may be linear, branched or cyclic. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and a tert-butyl group.
  • R 8 is preferably a hydrogen atom.
  • the number of carbon atoms of the alkyl group represented by R 9 is preferably 1 to 8, more preferably 1 to 6, from the viewpoint of suppressing the hydrophobicity of the solid phase carrier and expressing an appropriate hydrophobic interaction. 1 or 2 is particularly preferable.
  • the alkyl group may be linear, branched or cyclic.
  • the alkyl group preferably has no substituent such as a hydrophilic group.
  • T represents an integer of 1 to 5, preferably 1 to 3, and more preferably 1 from the viewpoint of suppressing the hydrophobicity of the solid phase carrier and expressing an appropriate hydrophobic interaction.
  • t is an integer of 2 to 5
  • t R 9 s may be the same or different.
  • alkyl vinyl benzene is particularly preferable.
  • content of component B 40 mass% or less is preferable with respect to all the monomer units in a polymer.
  • the content of component B is preferably 0.05% by mass or more, more preferably 0.5% by mass or more, based on all monomer units in the polymer, and also based on all monomer units in the polymer. More preferably, it is 35 mass% or less, More preferably, it is 30 mass% or less, Most preferably, it is 25 mass% or less. Even when the content of the component B is about 20% by mass, the desired effect of the present invention can be obtained.
  • the content ratio [(A) :( B)] of the component A and the component B in the polymer is 95: 5 to 50:50 by mass ratio.
  • the content ratio of component A exceeds 95 parts by mass, the brittleness of the porous carrier becomes high (particularly in the case of particles), and cracking tends to occur.
  • the content ratio of component A is less than 50 parts by mass, many alkyl groups derived from component B are present on the surface of the porous carrier, and impurities are easily adsorbed on the surface of the porous carrier.
  • the content ratio [(A) :( B)] is within the range of 95: 5 to 50:50, not only the cracking of the carrier is suppressed, but also when an antibody-binding ligand is used.
  • the content ratio [(A) :( B)] is preferably 90:10 to 55:45, more preferably 85:15 to 55:45, and 80:20 to 60:40 in terms of mass ratio. Is particularly preferred.
  • the said polymer contains the monomer unit derived from the monofunctional monomer except the (component C) component A and the component B further.
  • the content of component C is preferably 20 to 99.5% by mass with respect to all monomer units in the polymer. If it is in said range, the porous support
  • the content of component C is more preferably 25% by mass or more, further preferably 30% by mass or more, further preferably 40% by mass or more, and particularly preferably 50% by mass or more, based on all monomer units in the polymer.
  • it is preferably 95% by mass or less, more preferably 90% by mass or less, and particularly preferably 80% by mass or less, based on all monomer units in the polymer.
  • Component C includes (Component C-1) a monomer unit derived from an epoxy group-containing monovinyl monomer, and (Component C-2) a monomer unit derived from a monovinyl monomer not containing an epoxy group.
  • the polymer may contain one or more of these monomer units.
  • Component C-1 Monomer unit derived from epoxy group-containing monovinyl monomer
  • the epoxy group-containing monovinyl monomer is composed of one polymerizable vinyl group (group having an ethylenically unsaturated bond) and one or more in one molecule. And a monomer having an epoxy group.
  • the epoxy group-containing monovinyl monomer is a component for introducing an appropriate amount of an epoxy group to a solid phase carrier to obtain an appropriate ligand binding amount.
  • epoxy group-containing monovinyl monomer examples include glycidyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth) acrylate, ⁇ - (meth) acryl- ⁇ -glycidyl polyethylene Hydroxy group-free (meth) acrylic acid esters such as glycol; Hydroxy group-containing (meth) acrylic acid esters such as glycerin mono (meth) acrylate glycidyl ether; vinyl benzyl glycidyl ether, isopropenyl benzyl glycidyl ether, vinyl phenyl butyl In addition to aromatic monovinyl compounds such as glycidyl ether and vinylbenzyloxyethyl glycidyl ether, allyl glycidyl ether, 3,4-epoxy-1-butene, 3,4- And epoxy-3-methyl
  • epoxy group-containing monovinyl monomer epoxy group-containing (meth) acrylic acid esters and epoxy group-containing aromatic monovinyl compounds are preferable, epoxy group-containing (meth) acrylic acid esters are more preferable, and glycidyl (meta ) Acrylate and 4-hydroxybutyl (meth) acrylate glycidyl ether are more preferable, and glycidyl (meth) acrylate is particularly preferable.
  • epoxy group-containing aromatic monovinyl compounds vinylbenzyl glycidyl ether is preferable, and (4-vinylbenzyl) glycidyl ether is particularly preferable.
  • the epoxy group-containing aromatic monovinyl compound is preferably an epoxy group-containing styrene monovinyl compound, and specifically includes a compound represented by the formula (4).
  • R 4 represents a substituted or unsubstituted alkanediyl group having 1 or 2 carbon atoms
  • R 5 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms
  • R 6 represents an alkanediyl group having 2 or 3 carbon atoms
  • R 7 represents an alkyl group having 1 to 10 carbon atoms
  • m and n each independently represents an integer of 0 to 6
  • q represents an integer of 0 to 4.
  • the alkanediyl group represented by R 4 may be linear or branched.
  • a methane-1,1-diyl group is preferable from the viewpoint of improving the reactivity when binding an affinity ligand.
  • substituent that the alkanediyl group may have include a methyl group and an ethyl group.
  • R 5 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
  • the carbon number of the alkyl group represented by R 5 is preferably 1 or 2.
  • the alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group.
  • R 5 is preferably a hydrogen atom from the viewpoint of the reactivity of the polymerization reaction.
  • alkanediyl group represented by R 6 examples include ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2-diyl group, propane An ethane-1,1-diyl group and an ethane-1,2-diyl group are preferred.
  • the carbon number of the alkyl group represented by R 7 is preferably 1 to 6, more preferably 1 to 3, and further preferably 1 or 2 from the viewpoint of suppressing the hydrophobicity of the solid phase carrier. .
  • the alkyl group may be linear or branched. As an alkyl group, the same thing as the alkyl group shown by said R ⁇ 5 > is mentioned, for example.
  • n and n each independently represents an integer of 0 to 6, and m is preferably an integer of 0 to 3.
  • N is preferably an integer of 0 to 3, more preferably 0 or 1.
  • n R 6 s may be the same or different.
  • Q represents an integer of 0 to 4, and is preferably 0 or 1 from the viewpoint of suppressing the hydrophobicity of the solid phase carrier.
  • q R 7 may be the same or different.
  • the epoxy group-containing monovinyl monomer is preferably water-insoluble because the amount of ring-opening 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 Component C-1 is preferably 20 to 99.5% by mass with respect to all monomer units in the polymer. Within the above range, a porous carrier having excellent hydrophilicity and mechanical strength can be obtained.
  • the content of component C-1 is more preferably 25% by mass or more, further preferably 30% by mass or more, further preferably 40% by mass or more, and particularly preferably 50% by mass, based on all monomer units in the polymer. In addition, it is preferably 95% by mass or less, more preferably 90% by mass or less, and particularly preferably 80% by mass or less, based on all monomer units in the polymer.
  • Component C-2 Monomer unit derived from monovinyl monomer not containing epoxy group
  • Monovinyl monomer not containing an epoxy group has one polymerizable vinyl group (group having an ethylenically unsaturated bond) in one molecule. It is a vinyl monomer having no epoxy group.
  • the monovinyl monomer which does not contain an epoxy group will be described by being divided into a hydroxy group-free monovinyl monomer which does not contain an epoxy group and a hydroxy group-containing monovinyl monomer which 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 aromatic vinyl compounds such as styrene and ⁇ -methylstyrene.
  • hydroxy group-containing monovinyl monomer examples 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. It can be. Further, the number of hydroxy groups contained in the hydroxy group-containing monovinyl monomer not glycerol mono (meth
  • the monovinyl monomer not containing an epoxy group a hydroxy group-containing monovinyl monomer not containing an epoxy group is preferable, and glycerol mono (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, hydroxyethyl (Meth) acrylamide is more preferable, and glycerol mono (meth) acrylate and hydroxyethyl (meth) acrylamide are particularly preferable.
  • the content of component C-2 is preferably 40% by mass or less based on the total monomer units in the polymer.
  • the content of component C-2 is more preferably 35% by mass or less, still more preferably 30% by mass or less, and particularly preferably 20% by mass or less, based on all monomer units in the polymer.
  • the content of component C-2 may be 0% by mass, but when component C-2 is contained, it is preferably 5% by mass or more based on all monomer units in the polymer.
  • the porous carrier for affinity chromatography of the present invention desirably has a large surface area from the viewpoint of the processing capacity per unit time, and is preferably a porous body having a large number of pores of an appropriate size.
  • the form of the porous carrier for affinity chromatography of the present invention may be any of particulate, monolith, plate, fiber, membrane (including hollow fiber), and any form can be selected.
  • the water-insoluble carrier porous beads, monoliths or membranes are preferable.
  • the antibody affinity ligand arranged on the water-insoluble carrier and the alkyl group in Component B function in concert, a certain residence time is obtained.
  • the resulting porous beads (porous beads) are preferred.
  • the particle distribution having a circularity of 0.95 or more of the porous carrier for affinity chromatography of the present invention is preferably 60 to 100%, more preferably 70 to 100%, and particularly preferably 80 to 100%.
  • the volume average particle size of the porous carrier for affinity chromatography of the present invention is preferably 35 to 100 ⁇ m, more preferably 40 to 90 ⁇ m, and more preferably 40 to 80 ⁇ m from the viewpoint of the dynamic binding amount of the target and the pressure characteristics. Is particularly preferred.
  • the volume average particle diameter means a value measured by laser diffraction / scattered particle diameter distribution measurement or the like, and specifically means a value measured by the method described in Examples.
  • the coefficient of variation of the volume average particle diameter is preferably 40% or less, more preferably 30% or less.
  • the specific surface area of the porous carrier for affinity chromatography of the present invention is preferably 70 m 2 / g or more, more preferably 80 m 2 / g or more, and further preferably 90 m 2 / g or more. When the specific surface area is in the above range, the amount of dynamic binding of the target is high, and the ability to remove aggregates is excellent.
  • the volume average pore diameter of the porous carrier for affinity chromatography of the present invention is preferably 10 to 300 nm, more preferably 20 to 200 nm, from the viewpoint of the dynamic binding amount of the target substance and the ability to remove aggregates. ⁇ 100 nm is particularly preferred.
  • the coefficient of variation, specific surface area, and volume average pore diameter are values measured by laser diffraction / scattered particle diameter distribution measurement or the like.
  • the ligand-bound porous carrier of the present invention is obtained by binding an affinity ligand to the porous carrier for affinity chromatography of the present invention.
  • the ligand-bound porous carrier of the present invention is similar to the above-described porous carrier for affinity chromatography of the present invention in terms of particle distribution having a circularity of 0.95 or more, volume average particle size, coefficient of variation, specific surface area, and volume average pore size. Those within the range are preferred.
  • the affinity ligand in the present invention selectively collects (binds) a target (target) molecule from a set of molecules based on specific affinity between molecules represented by binding of an antigen and an antibody. Indicates substance.
  • the affinity ligand is not particularly limited as long as it specifically binds to a target.
  • proteins, nucleic acids, peptides, enzymes, chelate compounds, receptors, aptamers, antibodies, antigens, vitamins, metal ions, etc. Can be mentioned.
  • proteins, nucleic acids, peptides, enzymes, and chelate compounds are preferable, and proteins and peptides are more preferable.
  • an immunoglobulin binding protein is more preferable.
  • Antibody affinity ligands that can be used in the present invention are particularly limited as long as they have characteristics capable of specifically binding to Fc-containing molecules that are constant regions of antibodies, Fab-containing molecules that are variable regions of antibodies, or antibodies.
  • Peptide ligands, protein ligands, and chemically synthesized ligands (synthetic compounds) are preferred. From the viewpoint of specificity for the target molecule, a peptide or protein ligand is more preferred.
  • antibody affinity ligands include protein A, protein G, protein L, protein H, protein D, protein Arp, protein Fc ⁇ R, antibody-binding synthetic peptide ligand, camelid antibodies or fragments thereof, and related substances.
  • the antibody affinity ligand is not particularly limited as long as it has a target molecule binding domain (peptide monomer or protein, single domain), but is preferably a multimeric peptide or protein (multiple domain) in which two or more domains are linked, 2 to 10 are more preferable, 2 to 8, further 2 to 6 are preferable, and a multimeric protein in which 3 to 6 domains are linked is particularly preferable.
  • These multimeric proteins may be homopolymers such as homodimers and homotrimers that are linked bodies of a single target molecule-binding domain, and if the target molecules are the same, a plurality of types of target molecule-binding domains. It may be a heteropolymer such as a heterodimer or heterotrimer which is a linked body.
  • a method for linking the target molecule binding domain of the antibody affinity ligand a method that does not destabilize the three-dimensional structure of the multimeric protein is preferable.
  • a linking method via a terminal amino acid of the domain sequence, an amino acid residue of the domain sequence examples include a method of linking without intervening, a method of linking with amino acid residues other than one or a plurality of domain sequences, and are not limited to these methods.
  • fusion protein obtained by fusing a multimeric protein with another protein having different functions can be preferably used.
  • fusion proteins include proteins fused with albumin and GST (glutathione S monotransferase), nucleic acids such as DNA aptamers, drugs such as antibiotics, proteins fused with polymers such as PEG (polyethylene glycol), etc.
  • PEG polyethylene glycol
  • the affinity ligand binding amount is preferably 10 to 200 mg, more preferably 25 to 160 mg, still more preferably 30 to 140 mg, and particularly preferably 40 to 100 mg per 1 g of the dry weight of the porous carrier from the viewpoint of dynamic binding capacity. is there.
  • the affinity ligand binding amount may be measured by the same method as in Examples described later.
  • the amino group of the affinity ligand may be bound to the synthetic polymer support via a formyl group introduced on the carrier, and the amino group of the affinity ligand is activated on the synthetic polymer support. It may be bound to the carrier via a carboxy group, or the amino group of the affinity ligand may be bound to the carrier via an epoxy group on the synthetic polymer support.
  • the functional group introduced into the synthetic polymer support is not particularly limited as long as it is a functional group capable of forming a covalent bond with the affinity ligand.
  • an epoxy group epichlorohydrin
  • cyanogen bromide N, N— Hydroxyl group, aldehyde group or activating rubonic acid group activated by disuccinimidyl carbonate (DSC) or the like
  • DSC disuccinimidyl carbonate
  • NHS N monohydroxysuccinimide
  • CDI carbonyldiimidazole
  • the protein ligand can be immobilized on a carrier by reacting a part of the functional group of the protein with a part of the functional group of the carrier.
  • a side chain carboxy group and the like are exemplified, but not limited thereto.
  • a functional group for example, a linker or spacer
  • Charged amine Charged amine
  • a technique for immobilizing a proteinaceous ligand on an agarose carrier derivatized with an NHS activated carboxylic acid as a part of a linker arm US Pat. No. 5,260,373, JP 2010-133733.
  • Japanese Patent Laid-Open No. 2010-133734 Japanese Patent Laid-Open No. 2010-133734
  • the affinity ligand is accumulated on the support, and then an antibody affinity is formed on the support without forming a covalent bond between the associative group and the affinity ligand.
  • a method of individually immobilizing ligands has also been proposed (Japanese Patent Laid-Open No. 2011-256176), and this may be adopted.
  • the purification method of the present invention is characterized by using the ligand-bound porous carrier of the present invention.
  • the purification method of the present invention may be carried out in the same manner as in the conventional method except that the ligand-bound porous carrier of the present invention is used.
  • step A the ligand-binding porous carrier of the present invention is contacted with a target that can be captured by an affinity ligand, the dissociation solution that dissociates the affinity ligand and the target, And a step B of dissociating the target substance by contacting the carrier that has captured the target substance.
  • the target when the target is an antibody that easily aggregates, it may be necessary to selectively separate the monomer from a mixture of the target monomer and the target aggregate.
  • the target monomer is selectively separated by utilizing the difference in physical interaction between the surface of the ligand-bound porous carrier of the present invention and the target monomer or aggregate.
  • the ligand-bound porous carrier of the present invention can be used as a mixed mode carrier utilizing an affinity ligand and an alkyl group.
  • the step B is a stepwise method using two or more salt concentrations at which the ionic strength increases stepwise under the condition that the target substance is dissociated from the affinity ligand, or a salt concentration stepwise. It can be preferably exemplified that it is a step of dissociating by a gradient method (or a combination of both) for increasing the ratio. As a result, the monomer and the aggregate can be separated and eluted, although the separation ability is slightly reduced even if the ion strength is kept constant without changing.
  • the purification method of the present invention shows the separation characteristics of monomers and aggregates, and can shorten the two-step chromatographic operation of the affinity purification step and the purification step using ion exchange groups to one step.
  • the type and amount of liquid used, and further reduction in working time can be expected.
  • the purification method of the present invention is a narrow pH range (preferably pH 3 to 4, more preferably pH 3.1 to 3.9, still more preferably pH 3.2 to 3.) in which the target molecule from the antibody affinity ligand is dissociated. 8), wherein the ionic strength (preferably 10 to 500 mM, more preferably 15 to 400 mM, more preferably 20 to 350 mM, two or more salt concentrations that increase stepwise in the above range are used, It is possible to obtain an elution fraction with a high monomer content by setting a “gradient mode” using salt concentrations that increase in a gradient in the range.
  • the dissociation pH is far away from the isoelectric point of the target molecule, so there is no significant difference in the width of the dissociated ion intensity for each antibody, and the conditions for using various target molecules can be set within a narrow range. Can be expected to be possible. Furthermore, as an antibody affinity ligand, the dissociation pH range can be set narrower, and effective washing is possible by using alkaline CIP washing. Therefore, from the viewpoint of stable process construction, the modified protein A Use is preferred.
  • Examples of the target molecule to be purified in the present invention include immunoglobulin G and its analogs (including derivatives).
  • an Fc region that is a constant region of an immunoglobulin molecule Fc fusion proteins (Fc-containing molecules) formed by fusing other functional proteins or peptides are included. These are used as raw materials for antibody drugs.
  • the purification method of the present invention will be exemplified in the case where the target molecule is immunoglobulin G, but the present invention is not limited thereto.
  • the method for purifying a target according to the present invention includes a step A in which the ligand-bound porous carrier of the present invention is brought into contact with a target that can be captured by an affinity ligand. After adjusting the pH of the protein solution containing immunoglobulin G to be near neutral, the solution is passed through a column packed with the ligand-binding porous carrier of the present invention, and immunoglobulin G is passed through the affinity ligand. It is adsorbed specifically on the carrier.
  • the loading pH is preferably 6 or more, more preferably 6.3 or more and 9 or less, and even more preferably 6.5 or more and 8.5 or less.
  • preparation of ionic strength is not particularly required, and nonspecific adsorption can be further suppressed by increasing the ionic strength in advance.
  • the washing step an appropriate amount of a buffer solution in a condition range in which the affinity ligand functions is passed through to wash the inside of the column. That is, the preferable range of pH may be the same range (pH near neutrality) as that of the load, and for example, 6 or more is preferable.
  • the target molecule, immunoglobulin G is adsorbed on the ligand-bound porous carrier of the present invention. At this time, impurities may be effectively removed by ionic strength or composition optimization at a pH near neutral.
  • the ionic strength is preferably 0.2 M or more, and more preferably 0.5 M or more.
  • this purification method includes the step B
  • the step B may be any of a stepwise method, a gradient method, and a combination thereof as described above.
  • Step B the column is replaced with a buffer solution having a low ionic strength near neutrality to prepare for the development of an ionic strength-dependent dissociation function by a cation exchange group during dissociation.
  • the pH of the dissociation solution can be the dissociation pH of immunoglobulin G from the antibody affinity ligand. The pH is determined centering on separation conditions determined by the type of affinity ligand and immunoglobulin G.
  • the pH is preferably set between 2 and 6. However, in order to avoid acid modification of the target molecule, pH 3.0 or higher is more preferable, pH 3.3 or higher is more preferable, and pH 3.5 or higher is particularly preferable.
  • the pH is preferably 5.5 or less, more preferably 5.0 or less.
  • the dissociation pH is generally set between 3.5 and 4.0, but is not limited thereto.
  • the dissociation ionic strength depends on the introduction ratio of the antibody affinity ligand and the alkyl group derived from component B, and also on the amount of immunoglobulin G loaded per unit volume. Optimization points can be set easily.
  • the antibody dissociation from the ligand-bound porous carrier of the present invention prepared according to the present invention can be applied by either a salt concentration gradient method or a stepwise method. A wise method is preferred. Furthermore, in order to simplify the operation, it is preferable to set conditions that can achieve antibody recovery and high monomer content in one step.
  • Immunoglobulin G purified using the ligand-bound porous carrier of the present invention prepared according to the present invention exhibits high monomer selectivity and a high monomer content in the eluate.
  • the ligand-bound porous carrier of the present invention By using the ligand-bound porous carrier of the present invention, high-specificity affinity purification and improvement of the monomer content that can be achieved mainly by cation exchange chromatography, a single chromatographic operation while maintaining a high recovery rate. Therefore, it is possible to reduce the load on the subsequent process and contribute to the improvement of the yield of the entire process and the improvement of the monomer content. That is, according to the present invention, it is possible to contribute to the improvement in productivity and the purification of antibody drug manufacturing processes.
  • Example 1 (1) 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) is added to 448 g of pure water, heated and stirred to dissolve the polyvinyl alcohol, cooled, and sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 0 0.045 g was added and stirred to prepare an aqueous solution S.
  • PVA-217 manufactured by Kuraray Co., Ltd.
  • sodium dodecyl sulfate manufactured by Wako Pure Chemical Industries
  • a monomer composition comprising 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and 14.15 g of styrene (manufactured by Wako Pure Chemical Industries).
  • 3.63 g of divinylbenzene manufactured by Wako Pure Chemical Industries
  • 0.36 g of 1-ethyl-4-vinylbenzene manufactured by ChemSampCo
  • 14.15 g of styrene manufactured by Wako Pure Chemical Industries
  • 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.
  • porous carrier V1 The porous particles contained in this dispersion are referred to as “porous carrier V1”.
  • modified protein A (ReSpigen rSPA) is dispersed in 40 mL of 1.2 M sodium sulfate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A dispersion.
  • An epoxidized porous particle dispersion (corresponding to 1 g in terms of particle dry mass) was added to the A dispersion. The dispersion was shaken and stirred at 25 ° C. for 5 hours to fix Protein A to the 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. 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 filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W1”.
  • Example 2 In step (1) of Example 1, 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and 14 of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical) The steps (1) and (2) of Example 1 were performed except that a monomer composition consisting of .15 g was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. The same operation was performed to obtain a porous particle dispersion.
  • porous carrier V2 The porous particles contained in this dispersion are referred to as “porous carrier V2”. Thereafter, 0.15 g of modified protein A (ReSpigen rSPA) is dispersed in 40 mL of 1.2 M sodium sulfate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A dispersion. This protein A dispersion The above-mentioned porous particle dispersion (corresponding to 1 g in terms of dry particle weight) was added. The dispersion was shaken and stirred at 25 ° C. for 5 hours to fix Protein A to the particles.
  • modified protein A ReSpigen rSPA
  • 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. 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 filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W2”.
  • Example 3 In the step (1) of Example 1, 3.59 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 3.41 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company) 10 A monomer composition consisting of .94 g was dissolved in 29.39 g of diisobutylketone (manufactured by Mitsui Chemicals) to prepare a monomer solution. Thus, a porous particle dispersion was obtained. The porous particles contained in this dispersion are referred to as “porous carrier V3”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W3”.
  • Example 4 In the step (1) of Example 1, 6.29 g of divinylbenzene (Wako Pure Chemical Industries), 5.39 g of 1-ethyl-4-vinylbenzene (ChemSampCo) and glycidyl methacrylate (Mitsubishi Gas Chemical) 6 . Similar to steps (1) and (2) of Example 1 except that a monomer composition consisting of .29 g was dissolved in 29.39 g of diisobutyl ketone (Mitsui Chemicals) to prepare a monomer solution. Thus, a porous particle dispersion was obtained. The porous particles contained in this dispersion are referred to as “porous carrier V4”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W4”.
  • Example 5 In step (1) of Example 1, 0.90 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), 0.72 g of n-octyl acrylate (manufactured by Kyoeisha Chemical Co., Ltd.) and 16.33 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company) The same operation as in steps (1) and (2) of Example 1 except that a monomer composition was dissolved in 29.39 g of diisobutyl ketone (Mitsui Chemicals) to prepare a monomer solution. To obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as “porous carrier V5”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W5”.
  • Example 6 In Step (1) of Example 1, 6.41 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), 0.37 g of cyclohexyl methacrylate (manufactured by Kyoeisha Chemical Co., Ltd.) and 4-hydroxybutyl acrylate glycidyl ether (manufactured by Nippon Kasei Co., Ltd.) 11 Steps (1) and (2) of Example 1 except that a monomer composition consisting of .54 g was dissolved in 29.37 g of acetophenone (manufactured by Inoue Fragrance Co., Ltd.) and a monomer solution was prepared.
  • a monomer composition consisting of .54 g was dissolved in 29.37 g of acetophenone (manufactured by Inoue Fragrance Co., Ltd.) and a monomer solution was prepared.
  • porous carrier V6 The porous particles contained in this dispersion are referred to as “porous carrier V6”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W6”.
  • Example 7 In step (1) of Example 1, 3.62 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 0.91 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and glycerol monomethacrylate (manufactured by Mitsubishi Gas Chemical Company) The steps (1) and (2) of Example 1 except that the monomer composition consisting of 13.58 g was dissolved in 29.38 g of diisobutyl ketone (Mitsui Chemicals Co., Ltd.) to prepare a monomer solution. The same operation was performed to obtain a porous particle dispersion.
  • porous carrier V7 The porous particles contained in this dispersion are referred to as “porous carrier V7”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain protein A fixed particles in which unreacted epoxy groups were opened. Next, the protein A fixed particles in which the unreacted epoxy group was opened were 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). Unreacted epoxy groups were ring-opened by shaking and stirring at 25 ° C. for 17 hours.
  • 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 filler-containing liquid for affinity chromatography.
  • the filler contained in this liquid is referred to as “porous carrier W7”.
  • Example 8 In step (1) of Example 1, 2.70 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 1.80 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and (4-vinylbenzyl) glycidyl ether (Toray Industries, Inc.) A monomer composition consisting of 13.49 g of Fine Chemical Co., Ltd.
  • porous carrier V8 The porous particles contained in this dispersion are referred to as “porous carrier V8”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier W8”.
  • step (1) of Example 1 a monomer composition consisting of 5.49 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 12.80 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company) was converted into acetophenone (Inoue Fragrance Factory). Except that a monomer solution was prepared by dissolving in a mixed solution of 21.52 g (made by the company) and 7.35 g (made by Toyo Gosei Co., Ltd.). Operation was performed to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as “porous support X1”.
  • step (1) of Example 1 5.34 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 7.11 g of isobutyl methacrylate (manufactured by Kyoeisha Chemical Co., Ltd.) and 4-hydroxybutyl acrylate glycidyl ether (manufactured by Nippon Kasei Co., Ltd.) 34 g of the monomer composition was dissolved in a mixed solution of 21.50 g of acetophenone (Inoue Fragrance Co., Ltd.) and 7.34 g of 2-octanone (Toyo Gosei Co., Ltd.) to prepare a monomer solution.
  • acetophenone Inoue Fragrance Co., Ltd.
  • 2-octanone Toyo Gosei Co., Ltd.
  • porous particle dispersion The same operations as in steps (1) and (2) of Example 1 were performed to obtain a porous particle dispersion.
  • the porous particles contained in this dispersion are referred to as “porous support X2”.
  • protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography.
  • the filler contained in this liquid is referred to as “porous carrier Y2”.
  • step (1) of Example 3 In step (1) of Example 1, 6.29 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), 5.39 g of lauryl methacrylate (manufactured by Kyoeisha Chemical Co., Ltd.) and (4-vinylbenzyl) glycidyl ether (manufactured by Toray Fine Chemical Co., Ltd.) ) 6.29 g of the monomer composition was dissolved in 21.50 g of acetophenone (Inoue Fragrance Co., Ltd.) and 7.34 g of 2-octanone (Toyo Gosei Co., Ltd.) to prepare a monomer solution.
  • acetophenone Inoue Fragrance Co., Ltd.
  • 2-octanone Toyo Gosei Co., Ltd.
  • porous particle dispersion The porous particles contained in this dispersion are referred to as “porous support X3”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier Y3”.
  • step (1) of Example 1 6.41 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), 0.18 g of isobutyl methacrylate (manufactured by Kyoeisha Chemical Co., Ltd.) and (4-vinylbenzyl) glycidyl ether (manufactured by Toray Fine Chemical Co., Ltd.) ) 11.73 g of the monomer composition was dissolved in 21.50 g of acetophenone (Inoue Fragrance Co., Ltd.) and 7.34 g of 2-octanone (Toyo Gosei Co., Ltd.) to prepare a monomer solution.
  • acetophenone Inoue Fragrance Co., Ltd.
  • 2-octanone Toyo Gosei Co., Ltd.
  • porous particle dispersion The porous particles contained in this dispersion are referred to as “porous support X4”. Thereafter, protein A was fixed, unreacted epoxy groups were opened, and washed in the same manner as in Example 2 to obtain a filler-containing liquid for affinity chromatography. The filler contained in this liquid is referred to as “porous carrier Y4”.
  • Test Example 1 (particle size measurement) Laser diffraction scattering type particle size distribution measuring device (LS13320, manufactured by Beckman Coulter, Inc.) for the average particle diameters of porous carriers V1 to V8, W1 to W8, X1 to X4, and Y1 to Y4 obtained in each Example and Comparative Example It was measured by. The results are shown in Tables 1 and 2. In addition, the numerical value in a table
  • surface is the value which rounded off after the decimal point.
  • Test Example 2 Evaluation of circularity distribution
  • the porous carriers V1 to V8, W1 to W8, X1 to X4, and Y1 to Y4 obtained in each example and comparative example were adjusted to a solid content concentration of 10% by mass with pure water, and a quantitative control diaphragm pump (manufactured by Takumina) ) was used for 60 circulation processes at a discharge rate of 6 L / min. Thereafter, the porous carrier after the circulation treatment is diluted with water so that the solid content concentration is about 2.5% by mass, and the total count is obtained using a flow type particle image analyzer (model number FPIA-3000) manufactured by Sysmex Corporation.
  • Test Example 3 Quantification of protein A binding amount
  • the amount of protein A, which is a ligand bound to the porous carriers W1 to W8 and Y1 to Y4, obtained in each Example and Comparative Example was quantified using a bicinchoninic acid (BCA) reagent. Specifically, 1 mg of the porous carrier in terms of solid content was collected in a test tube, and the binding amount of protein A was quantified with BCA Protein Assay Kit of 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 Tables 1 and 2.
  • Test Example 4 Measurement of DBC Using GE Healthcare's AKTAprime plus, each of Examples and Comparative Examples of porous carriers W1 to W8 and Y1 to Y4 for a protein (human IgG antibody, Equitech Bio HGG-1000) at a linear flow rate of 300 cm / hr DBC was measured.
  • DBC was determined from the amount of protein captured during the through and the column packing volume, and evaluated according to the following criteria. The results are shown in Tables 1 and 2.
  • (DBC evaluation criteria) 35 mg / mL or higher: Good Less than 35 mg / mL: Poor
  • Test Example 5 Quantification of HCP and aggregates
  • Column columns (Tricorn 10/50 column manufactured by GE Healthcare) were packed with porous carriers W1 to W8 and Y1 to Y4 of each Example and Comparative Example 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.
  • HCP Host Cell Protein
  • Test Example 6 Measurement of consolidation line flow rate
  • the porous carriers V1 to V8, W1 to W8, X1 to X4, and Y1 to Y4 obtained in each Example and Comparative Example were packed into a column container so that the inner diameter was 16 mm and the packing height was 100 mm. It connected to AKTA pilot made by Healthcare Bioscience. Subsequently, the flow of pure water was started at a linear flow rate of 100 cm / hr, and the linear flow rate was gradually increased by 50 cm / hr every minute, and the flow rate was reduced to 0. The linear flow rate when a pressure increase with time exceeding 05 MPa was observed was recorded as the consolidation flow rate.
  • Each symbol in Tables 1 and 2 means the following compound.
  • DVB divinylbenzene
  • TMP trimethylolpropane trimethacrylate
  • EVB 1-ethyl-4-vinylbenzene
  • OCAC n-octyl acrylate
  • CHMA cyclohexyl methacrylate
  • IBMA isobutyl methacrylate
  • LauMA Lauryl methacrylate
  • C ST: Styrene
  • GMA Glycidyl methacrylate
  • HBAGE 4-hydroxybutyl acrylate glycidyl ether
  • GLM Glycerol monomethacrylate
  • VBGE (4-vinylbenzyl) glycidyl ether

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un support poreux pour chromatographie d'affinité et qui présente un excellent équilibre entre dureté et ténacité. Le support poreux pour chromatographie d'affinité comprend un polymère ayant une unité de monomère (composé A) dérivée d'un monomère polyvinylique et une unité de monomère (composé B) ayant un groupe alkyle ayant de 1 à 8 atomes de carbone dans sa chaîne latérale, et est caractérisé en ce que le rapport massique [(A):(B)] entre le composé A et le composé B contenus dans le polymère est de 95:5 à 50:50 et la teneur du composé A est de 0,5 à 40 % en masse par rapport à la totalité des unités de monomères dans le polymère.
PCT/JP2017/007172 2016-02-26 2017-02-24 Support poreux pour chromatographie d'affinité, support poreux conjugué à un ligand, procédé de purification de cible, et anticorps WO2017146225A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016-035622 2016-02-26
JP2016035622A JP2019070528A (ja) 2016-02-26 2016-02-26 多孔質担体、アフィニティクロマトグラフィー用多孔質担体、標的物の精製方法、及び抗体

Publications (1)

Publication Number Publication Date
WO2017146225A1 true WO2017146225A1 (fr) 2017-08-31

Family

ID=59686612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/007172 WO2017146225A1 (fr) 2016-02-26 2017-02-24 Support poreux pour chromatographie d'affinité, support poreux conjugué à un ligand, procédé de purification de cible, et anticorps

Country Status (3)

Country Link
JP (1) JP2019070528A (fr)
TW (1) TW201731592A (fr)
WO (1) WO2017146225A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008505851A (ja) * 2004-02-27 2008-02-28 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 抗体精製
WO2015199196A1 (fr) * 2014-06-27 2015-12-30 Jsr株式会社 Support pour chromatographie d'affinité
WO2016013609A1 (fr) * 2014-07-25 2016-01-28 旭化成メディカル株式会社 Vecteur de chromatographie d'échange d'ions positifs et son procédé d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008505851A (ja) * 2004-02-27 2008-02-28 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 抗体精製
WO2015199196A1 (fr) * 2014-06-27 2015-12-30 Jsr株式会社 Support pour chromatographie d'affinité
WO2016013609A1 (fr) * 2014-07-25 2016-01-28 旭化成メディカル株式会社 Vecteur de chromatographie d'échange d'ions positifs et son procédé d'utilisation

Also Published As

Publication number Publication date
TW201731592A (zh) 2017-09-16
JP2019070528A (ja) 2019-05-09

Similar Documents

Publication Publication Date Title
KR102410991B1 (ko) 친화성 크로마토그래피용 담체
JP6630036B2 (ja) 標的物の精製方法、及び、ミックスモード用担体
US9162161B2 (en) Filler for affinity chromatography
Jain et al. Protein purification with polymeric affinity membranes containing functionalized poly (acid) brushes
KR101354476B1 (ko) 크로마토그래피 매질 성능을 향상시키기 위한 그라프팅 방법
US9090665B2 (en) Filler for affinity chromatography
KR101921392B1 (ko) 고상 담체, 고상 담체의 제조 방법, 친화성 정제용 담체, 친화성 크로마토그래피용 충전제의 제조 방법, 친화성 크로마토그래피용 충전제, 크로마토그래피 칼럼 및 정제 방법
JP2019034963A (ja) 陽イオン交換基を用いた新規抗体精製法（Ｎｏｖｅｌ Ａｎｔｉｂｏｄｙ Ｐｕｒｉｆｉｃａｔｉｏｎ ｍｅｔｈｏｄ ｕｓｉｎｇ Ｃａｔｉｏｎ Ｅｘｃｈａｎｇｅｒ）
JP6335785B2 (ja) ミックスモード抗体アフィニティー分離マトリックスとそれを用いた精製方法および標的分子
JPS63307829A (ja) 光学異性体用分離剤
WO2015119255A1 (fr) Support en phase solide, procédé de production de support en phase solide, support pour purification par affinité, charge, colonne de chromatographie et procédé de purification
JP2022184990A (ja) バイオセパレーションのための複合材料
KR102549800B1 (ko) 크로마토그래피용 담체, 리간드 고정 담체, 크로마토그래피 칼럼, 표적 물질의 정제 방법 및 크로마토그래피용 담체의 제조 방법
EP2986625A2 (fr) Ligands en mode mixte
JPWO2007123242A1 (ja) ＩｇＧ精製用分離剤、及びそれを用いたＩｇＧ単量体の精製方法
WO2017146225A1 (fr) Support poreux pour chromatographie d'affinité, support poreux conjugué à un ligand, procédé de purification de cible, et anticorps
JP6602533B2 (ja) 担体の製造方法、担体、クロマトグラフィーカラム、及び目的物質の精製方法
JPH013129A (ja) 光学異性体用分離剤
JP7344232B2 (ja) バイオセパレーションのための複合材料
JP2020002079A (ja) 分離剤の製造方法、分離剤の洗浄方法及び抗体の精製方法
WO2023064949A1 (fr) Ligands de chromatographie par échange de cations en mode mixte à base de structures d'acide acétique à substitution 2-benzamido
JPH03251544A (ja) 光学異性体用分離剤

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17756659

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17756659

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP