EP1194228A1 - Materiau a affinite controlee avec polymere sensible a un stimulus et procede de separation/purification faisant intervenir ce materiau - Google Patents

Materiau a affinite controlee avec polymere sensible a un stimulus et procede de separation/purification faisant intervenir ce materiau

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Publication number
EP1194228A1
EP1194228A1 EP00927741A EP00927741A EP1194228A1 EP 1194228 A1 EP1194228 A1 EP 1194228A1 EP 00927741 A EP00927741 A EP 00927741A EP 00927741 A EP00927741 A EP 00927741A EP 1194228 A1 EP1194228 A1 EP 1194228A1
Authority
EP
European Patent Office
Prior art keywords
affinity
stimulus
substance
target substance
controlling material
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00927741A
Other languages
German (de)
English (en)
Inventor
Kimihiro Yoshizako
Yoshikatsu Akiyama
Teruo Okano
Katsuhiko Ueno
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GE Healthcare Japan Corp
National Institute of Advanced Industrial Science and Technology AIST
Original Assignee
Agency of Industrial Science and Technology
Japan Chemical Innovation Institute
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 Agency of Industrial Science and Technology, Japan Chemical Innovation Institute filed Critical Agency of Industrial Science and Technology
Publication of EP1194228A1 publication Critical patent/EP1194228A1/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • 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
    • 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
    • B01J20/3204—Inorganic carriers, supports or substrates
    • 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
    • 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
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • 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
    • 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
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • 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
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • 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
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • 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
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3265—Non-macromolecular compounds with an organic functional group containing a metal, e.g. a metal affinity ligand
    • 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
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • 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
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G01N2030/528—Monolithic sorbent material
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature

Definitions

  • This invention relates to an affinity-controlling material comprising (a) a stimulus-responsive polymer and (b) an affinitive substance (ligand) having affinity for a target substance that is present in a solution in contact with the material.
  • a stimulus for instance a physical stimulus
  • the chemical and/or physical environment provided around the ligand can be changed thereby changing the affinity between the ligand and the target substance.
  • the change in environment is typically related to a conformational change in the stimulus-responsive polymer.
  • solution means liquids, typically containing dissolved buffer components and salts ( ions ) .
  • Typical solutions have as the liquid component : a solvent such as water, an organic solvent and mixtures thereof.
  • Organic solvents include in particular those that are miscible with water, for example, water-miscible alcohols, acetonitrile, tetrahydrofuran , etc and mixtures thereof.
  • change in affinity “changing the affinity” etc refer to the apparent affinity, i.e. the affinity that can be measured under the conditions applied.
  • Affinity-controlling material will further on also be called separation medium or separation material.
  • the present invention further relates to an affinity- controlling material with which a desired target substance (metal ions, drugs, biological components, etc.) can be removed or separated and/or purified from mixtures containing other substances .
  • the present invention furthermore relates to a method for separating and purifying target substances (metal ions, drugs, biological components, etc. ) with the use of the above-mentioned affinity-controlling material.
  • the method preferably comprises keeping at least one condition other than temperature (for example, pH value of solution, organic solvent concentration or salt concentration) constant.
  • Separation and/or purification of a target substance from a mixture of substances by affinity chromatography and other adsorption based separation techniques typically encompass a binding step (adsorption step) and a release step (desorption or eluting step) .
  • the release step typically requires a change in the composition of the liquid in contact with the separation medium.
  • Illustrative examples for accomplishing the appropriate change are adding an organic solvent to a mobile phase, elevating the salt concentration of the mobile phase, or changing the pH value of the mobile phase. This also applies to the turbulent or non-turbulent liquid phases used in batch-wise procedures. These operations result in an increased risk for inactivation of physiologically active substances.
  • Galaev et al J. Chromatog. A 684 (1994) 37-43 and WO 94/154951 describe temperature elution of a target substance in a chromatographic system in which a plurality of ligand groups is covalently attached to a base matrix.
  • a temperature responsive polymer is indirectly affinity bound to the ligand groups by multi-point attachment, i.e. the attachment of the thermo- responsive polymer is depending on the prior attachment of the ligands to the base matrix. By changing the temperature the ligand becomes more or less prone to affinity bind to its target substance.
  • thermoresponsive polymer for the separation of biomolecules .
  • the matrices may be porous.
  • the hydrophobic groups utilized are inherent in the polymer as such. There is no ligand that has been covalently attached to the polymer after polymerisation.
  • Separation media are often in the form of particles that may be porous and non-porous.
  • the particles typically comprise a base matrix to which the ligand is attached directly or indirectly, for instance via a spacer.
  • the particle material may be a synthetic polymer such as crosslinked polymerisates of styrenes , acrylates/methacrylates and the like.
  • polystyrene particles and polymethacrylate particles per se are relatively hydrophobic and therefore often exhibit a pronounced non-specific adsorption of various substances that may be present together with a desired target substance.
  • the objects of the invention are to provide solutions to the problems discussed above thereby enabling improved separation methods and separation materials.
  • an affinity-controlling material/separation medium can be synthesized by attaching a stimulus-responsive polymer and an affinitive substance (ligand) of a target substance independently to a support, i.e. via separate linking structures.
  • a target substance adsorbed by the above-mentioned affinity- controlling material can be desorbed under a physical stimulus, such as a temperature change, while keeping at least one conditions other than the stimulus concerned constant. If, for instance, the stimulus is a temperature change, the condition(s) to be kept constant may be selected amongst, for example, pH, organic solvent concentration, salt concentration etc.
  • the present inventors have furthermore found that the bonding ability between a ligand and a target substance depends on the length of a spacer by which the ligand is attached to a support/base matrix or on the size of the stimulus-responsive polymer bonded to the support .
  • This embodiment also includes to chemically treat the particles with acidic or basic substances in order to introduce hydrophilic groups and/or groups that will enable covalent attachment of the ligand and/or the stimulus-responsive polymer, for instance hydroxy and/or amino groups.
  • the chemical treatment requires that the starting material (monomer) used in the membrane emulsification method exhibits a reactive group that is able to react with the acidic or basic substance utilized. Typical reactive groups have been epoxy groups. The present invention has been completed based on these findings .
  • the present invention relates to an affinity-controlling material/separation medium of the type defined in the introductory part .
  • One of the major characteristic features is that the stimulus-responsive polymer and the ligand are attached by separate/independent links to a base matrix.
  • the stimulus may be a physical stimulus and is in the experimental part typified by a temperature change.
  • the present invention further relates to an affinity- controlling material wherein the affinity of an affinative substance of a target substance is reversibly changed by changing the chemical or physical environment of a stimulus-responsive polymer under a physical stimulus while keeping at least one condition other than the changed condition constant, i.e. subjecting the thermo-responsive polymer to a change in one physical stimulus while keeping at least one of the other stimulus constant. If the changed stimulus is the temperature, then at least one condition/stimulus except the temperature is maintained essentially constant (for example, pH, organic solvent concentration or salt concentration) .
  • the present invention furthermore relates to an affinity-controlling material/separation medium as defined above wherein the affinity between the ligand and the target substance depends on (a) the length of the spacer attaching the ligand to the base matrix or (b) the size, for instance as reflected in molecular weight, of the stimulus-responsive polymer.
  • the present invention further relates to an affinity- controlling material/a separation medium as defined above wherein the support (base matrix) comprises hydrophilic porous polymer particles preferably having a uniform particle size and/or being produced by polymerisation of a monomer emulsion/suspension obtained from the membrane emulsification method. Hydrophobic particles exhibiting functional groups that are reactive with acidic or basic substances, may be rendered less hydrophobic by reaction with this kind of substances.
  • Typical examples of reactive groups are epoxy groups, i.e. if the membrane emulsification method involves polymerisation the starting monomer contains an epoxy group.
  • An additional embodiment of the invention is a method for the separation of one or more target substances from a liquid sample (solution) (liquid I). This embodiment comprises the steps of
  • step (a) bringing a liquid sample (liquid (I)) containing a target substance in contact with a separation medium (including a chromatographic packing) which is functionalized with a ligand which is capable of affinity binding to the target substance, said contact being under conditions permitting binding of said target substance to said ligand; (b) contacting said carrier with a liquid (II) not containing said target substance under conditions such that the target substance is released from said ligand to liquid (II).
  • the liquid sample is preferably separated from the separation medium.
  • liquid (II) may be separated from the separation medium.
  • the target substance may, if so desired, be worked up from liquid II.
  • the separation medium may be washed between step (a) and step (b).
  • the liquids typically have been aqueous for target substances that are biologically and/or physiologically active molecules, e.g. bioorganic molecules having structures selected amongst nucleotide structure (including nucleic acids), polypeptide structure (including proteins), carbohydrate structure, steroid structure etc.
  • target substances that are biologically and/or physiologically active molecules, e.g. bioorganic molecules having structures selected amongst nucleotide structure (including nucleic acids), polypeptide structure (including proteins), carbohydrate structure, steroid structure etc.
  • This embodiment of the invention is characterized in that (i) said separation medium comprises a support/base matrix to which a stimulus-responsive polymer as defined elsewhere in this specification and the ligand are linked separately, and (ii) subjecting in step (a) and at least during binding of the target substance to the ligand, the separation media to a stimulus at a level/intensity at which the stimulus-responsive polymer is in a conformation enhancing binding of the target substance to the ligand, and
  • step (iii) subjecting in step (b) and at least during release of the target substance from the ligand, the separatory material to a stimulus at a level/intensity at which the stimulus-responsive polymer is in a conformation hindering binding of the target substance to the ligand.
  • step (a) Compared to step (a), the level/intensity of the stimulus in step (b) is on the opposite side of the critical level/intensity for the stimulus-sensitive polymer used.
  • the process can be made cyclic in case step a is repeated after step b, typically after separate washing/regeneration steps and equilibration steps.
  • Various embodiments of the inventive method may be carried out in a batch-wise or a chromatographic mode.
  • Chromatographic modes may be carried out by permitting the various liquids in plug flow (mobile phase) to pass through a bed of the separation medium while subjecting the bed to the appropriate stimulus for the individual steps and the stimulus-responsive polymer that is attached to the base matrix.
  • the bed may be a porous monolith or a bed of packed or fluidised particles.
  • Batch-wise modes concern suspended particles in combination with turbulent flow and/or turbulent liquids.
  • Fig. 1 provides a chromatogram showing control of the affinity of BSA by BC-10 with temperature change.
  • the physical stimulus to be used in the present invention is exemplified by temperature.
  • stimulus-responsive polymer used other stimulus may apply, for instance, light, magnetic field, electrical field, pH etc.
  • Stimulus-responsive polymers are often called "intelligent polymers”.
  • Stimulus-responsive polymers are characterized in that they upon being subjected to the correct kind and intensity or level of a stimulus undergo a conformational and reversible change of their physico-chemical properties. The change may be a switch from a pronounced hydrophobicity to a pronounced hydrophilicity or vice versa.
  • the exact level/intensity the required stimulus at which the switch occurs is called critical level or critical intensity of the stimulus and will depend on the structure of the polymer and often also on other conditions (solvent, solutes such as salts etc) .
  • the most wellknown and most utilized polymers of this kind respond to heat (thermo- responsive or temperature-responsive polymers).
  • Temperature- responsive polymers are recognized by a sharp temperature limit at which they switch from a pronounced hydrophilic state to a pronounced hydrophobic state and vice versa.
  • CST critical solution temperature
  • LCST lower critical solution temperature
  • UCST upper critical solution temperature
  • the stimulus-responsive polymer to be used in the invention preferably has an insignificant affinity for the target substance compared to the affinity between the target substance and the ligand attached to the support. Preferably there is no significant affinity between the ligand and the stimulus-responsive polymer.
  • the stimulus-responsive polymer to be used in the present invention include poly(N-substituted acrylamide) such as poly(N-isopropyl acrylamide), poly(N-substituted methacrylamide) such as poly(N-isopropyl methacrylamide ) , poly(N,N-disubstituted acrylamide), poly(N,N-disubstituted methacrylamide), polymethyl vinyl ether, poly(ethylene oxide-propylene oxide) copolymer, polyvinyl alcohol derivatives typified by partly saponified polyvinyl alcohol and cellulose derivatives typified by methyl cellulose. It is also possible to introduce reacting functional groups (for example, amino, carboxyl or hydroxyl groups
  • Ligands may be attached to the base matrix via affinity bonds or via covalent bonds, preferably the latter. According to the present inventive concept it is not via the stimulus- responsive polymer.
  • One typical kind of ligands affinity binds to the target substance by more or less pure ionic (electrostatic) interactions. Alternatively the binding includes more complex interactions such as in conventional affinity binding (affinity adsorption).
  • the ligands comprise positively or negatively charged entities (ion exchange; the immobilised entity being selected among primary, secondary, tertiary and quaternary ammonium, sulphonate, sulphate, phosphonate, phosphate, carboxy etc groups). More complex interactions are illustrated by the ligand being an individual affinity member in the pairs ,
  • Affinity members also include entities participating in catalytic reactions, for instance enzymes, enzyme substrates, cofactors, cosubstrates etc. Members of cell-cell and cell- surface interactions and a synthetic mimetics of bioproduced affinity members are also included.
  • the term ligand also includes more or less complex organic molecules that binds through affinity to complex biomolecules , for instance having oligo or polypeptide structure (including proteins), oligo and polynucleotide structure (including nucleic acids), oligo- or polysaccharide structures etc.
  • ligands to be used in the present invention include dyes such as CIBACRONE BLUE F3G-ATM (manufactured by Fluka) and other complex dyes, iminodiacetic acid, sugar chains such as glucose, proteins such as heparin and lectin, biotin, benzamidine, lysine, arginine, peptides and DNA. It is also according to the invention possible to control the bonding ability of the target substance by covalently bonding the affinitive substance of the target substance to the support via a spacer such as a bivalent alkyl group or an ethylene oxide grou .
  • a spacer such as a bivalent alkyl group or an ethylene oxide grou .
  • the base matrix e.g. chromatographic packings
  • the separation medium to be used in the inventive method comprises a base matrix (carrier) which may be based on organic and/or inorganic material.
  • the base matrix is preferably hydrophilic .
  • the base matrix is preferably based on a polymer, which preferably is insoluble and more or less swellable in water, preferably to a gel .
  • Hydrophobic polymers that have been derivatized to become hydrophilic are included in this definition. Suitable polymers are polyhydroxy polymers, e.g. based on polysaccharides , such as agarose, dextran, cellulose, starch, pullulan, etc.
  • polyacrylic acid amide polymethacrylic acid amide
  • polymethacrylic acid amide poly(hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylates ) and polymethacrylates (e.g. polyglycidylmethacrylate)
  • polyvinylalcohols and polymers based on styrenes and divinylbenzenes and copolymers in which two or more of the monomers corresponding to the above-mentioned polymers are included.
  • Polymers, which are soluble in water may be derivatized to become insoluble, e.g. by cross-linking and by coupling to an insoluble body via adsorption or covalent binding.
  • Hydrophilic groups can be introduced on hydrophobic polymers (e.g. on copolymers of monovinyl and divinylbenzenes) by polymerization of monomers exhibiting groups which can be converted to OH, or by hydrophilization of the final polymer, e.g. by adsorption of suitable compounds, such as hydrophilic polymers .
  • Suitable inorganic materials to be used in base matrices are silica, zirconium oxide, graphite, tantalum oxide etc.
  • Preferred base matrices lack groups that are unstable against hyrolysis, such as silan, ester, amide groups and groups present in silica as such.
  • Preferred base matrices contain functional groups that can be used for attaching covalently the stimulus-responsive polymer and/or the ligand. This kind of functional groups are illustrated by hydroxy, carboxy, amino groups etc.
  • the matrix may be porous or non-porous. This means that the matrix may be fully or partially permeable (porous) or completely impermeable to the compound to be removed (non- porous) .
  • the pores may have sizes > 0.1 ⁇ m, such as ⁇ 0.5 ⁇ m, by which is meant that a sphere ⁇ 0.1 ⁇ m respective ⁇ 0.5 ⁇ m in diameter is able to pass through.
  • An applied liquid may be able to flow through this kind of pore system (convective pore system).
  • the ratio between the pore sizes of the convective pore system and the diameter of the particles typically is in the interval 0.01-0.3, with preference for 0.05-0.2.
  • Pores having sizes ⁇ 0.1 ⁇ m, such as ⁇ 0.5 ⁇ m, are often called macropores .
  • the base matrix may also have pores with sizes ⁇ 0.5 ⁇ m, such as ⁇ 0.1 ⁇ m by which is meant that only spheres with diameters ⁇ 0.5 ⁇ m, such as ⁇ 0.1 ⁇ m, can pass through. Pores having sizes ⁇ 0.5 ⁇ m, such as ⁇ 0.1 ⁇ m, are often called micropores .
  • the base matrix is in the form of irregular or spherical particles with sizes in the range of 1-1000 ⁇ m, preferably 5-50 ⁇ m for high performance applications and 50-300 ⁇ m for preparative purposes.
  • Particles to be used may be monodisperse (monosized) or polydispersed (polysized) .
  • monodispersed particles is meant a particle populations having more than 95% of the particles with sizes within their mean diameter ⁇ 5%, which in the context of the present invention contemplate the expression particles of uniform size.
  • Polydispersed particles encompass other populations of particles.
  • the base matrix may also be in form of a monolith having at least macropores as defined above.
  • Alternative geometric forms are the interior walls of tubes and the like
  • the stimulus responsive polymer and the ligand as defined above may be attached to the outer surfaces and/or on the interior surfaces (macropore and/or micropore surfaces) of the base matrix.
  • the stimulus responsive polymer and the ligand may be attached to the base matrix by physical adsorption and/or covalent attachment, preferably the latter. It is particularly preferable to use as the support/base matrix hydrophilic porous polymer particles having a uniform particle size, which are produced by the membrane emulsification method followed by a chemical treatment with an acidic substance or a basic substance as discussed above.
  • the membrane emulsification method as used in the present invention is a method which comprises passing a first liquid through a glass membrane, preferably made of glass , into a second liquid which is mot miscible with the first liquid, thus forming droplets of an essentially size in the second liquid.
  • This method is described in, for example, S. Omi, K. Katami, A. Yamamoto and M. Iso. J. Appl. Polym. Sci . , 51 (1994) 1-11.
  • the first liquid contains a polymerizable monomer and the droplets are subjected to polymerization, particles will form in the second liquid.
  • the preferred the support materials (base matrices) according to the inventor's novel finding is produced in the following manner: A liquid mixture (first liquid) is prepared from a monomer, which serves as the starting material for polymer particles, and a diluent, etc. Next, one side of a porous glass membrane is filled with this liquid mixture and the opposite side with a second liquid which is not miscible with the first liquid. Pressure is the applied to the liquid mixture so that it passes through the membrane and forms droplets in the second liquid.
  • the first liquid may be immiscible with water and the second liquid aqueous containing an emulsion stabilizer, etc . to give an emulsion consisting of droplets of an essentially uniform size.
  • polymerization is carried out by, for example, heating to thereby give latex particles having a uniform particle size .
  • the monomer contains a group reactive with an acidic or basic substance
  • the latex particles can be stirred in a solution containing this kind of substances to give hydrophilic porous polymer particles .
  • reactive functional groups such as amino groups can be introduced into the support .
  • the stimulus-responsive polymer or the ligand can be covalently attached to the support.
  • Examples of the monomer to be used in producing the hydrophilic porous polymer particles having a uniform particle size include glycidyl acrylate, glycidyl methacrylate , diacrylates (for example, ethylene diacrylate) , dimethacrylates ( for example , ethylene dimethacrylate) , glycidyl vinylbenzyl ether and divinylbenzene. It is also possible to combine these monomers .
  • the diluent to be used in the production of the hydrophilic porous polymer particles having a uniform particle size may be an arbitrary compound as long as it is not polymerizable with the monomers used. Examples thereof include aromatic solvents/compounds , such as toluene and aliphatic compounds such as dodecane .
  • Examples of the acidic substance or basic substance to be used in the production of the hydrophilic porous polymer particles having a uniform particle size include sulfuric acid, hydrochloric acid, nitric acid, acetic acid, sodium hydroxide, ammonia and aliphatic diamines such as 1 , 6-diaminohexane.
  • the affinity-controlling material/separation material according to the present invention can be produced by, for example ,
  • (c) a method which comprises covalently attaching the affinitive substance of the target substance and the stimulus-responsive polymer or a copolymer thereof respectively to the support at the same time.
  • the molecular weight of the obtained polymer was determined by gel permeation chromatography (GPC) and end-group analysis .
  • GPC gel permeation chromatography
  • end-group analysis was performed using dimethylformamide containing 10 mM lithium bromide as a mobile phase, a column a-3000 (TOSOH Co., Japan) column, and polystyrene as a standard reference material .
  • the number average molecular weight and the weight average molecular weight of the synthesized poly(N- isopropylacrylamide) were found to be about 4,500 and 10,000, respectively.
  • the carboxyl terminal groups of the synthesized poly(N-isopropylacrylamide) were determined by end group analysis with a COIN sodium hydroxide solution. As a result, the number average molecular weight was about 5000.
  • the number average molecular weight of the synthesized poly(N-isopropylacrylamide) determined by GPC is essentially the same as that determined by end group analysis .
  • the synthesized poly(N-isopropylacrylamide) ( 10 g) , N- hydroxysuccinimide (0.25 g) , and N,N' -dicyclohexyl- carbodiimide (0.45 g) were dissolved in tetrahydrofuran (60 ml) , and the resulting solution was stirred at room temperature for 12 hours. The resulting precipitate was collected by filtration and reprecipitated in diethyl ether to give poly(N- isopropylacrylamide) whose carboxyl group at one end is esterified with N-hydroxysuccinimide.
  • the emulsion was subjected to polymerization at 70°C for 6 hours, and the latex particles with a uniform particle size were in a high yield.
  • the average particle diameter of the latex particles was 12.5 ⁇ m, the CV (coefficient of variation)value was 12.4%, and the particles were uniform in size.
  • the synthesized latex particles (3.5 g) were dispersed into an aqueous solution (160 ml) containing 1 , 6-hexyldiamine (1.8 g) , and the mixture was stirred at 30°C for 2 hours.
  • the hydrochloric acid-calcium chloride method (Nakamura etal., Kobunshi Ronbunshu, 38(7) (1981) 485-491.) gave that the latex particles had 3.1 mmol/g of epoxy groups on their surfaces prior to the treatment with 1 , 6-hexyldiamine. Furthermore, the assay using titration revealed that 0.36 mmol/g of amino groups were introduced onto the surfaces of the hydrophilic porous polymer particles by the treatment with 1 , 6-hexyldiamine.
  • the 1 , 6-hexyldiamine-treated hydrophilic porous polymer particles (3.5 g) were then added to 10 ml of acetic anhydride, the solution was stirred to acetylate the amino groups, thereby obtaining amidated particles .
  • These amidated particles did not adsorb bovine serum albumin (BSA) in a 20 mM phosphate buffer (pH 7.0) used as a mobile phase. This indicates that the amidated hydrophilic porous polymer particles had a hydrophilicity sufficient to render them suitable as a chromatography carrier (base matrix) protein separation by affinity chromatography.
  • BSA bovine serum albumin
  • the packing material was prepared by acetylating the remaining amino groups in the support with acetic anhydride.
  • the unreacted amino groups of the support were acetylated by adding acetic anhydride (0.11 ml) to the suspension followed by stirring at 30°C for 1 hour.
  • the resulting support on which the spacer and poly(N-isopropylacrylamide) are immobilized was washed with acetonitrile and acetone, and then dried at room temperature.
  • BSA was allowed to be adsorbed at 40°C by Cibacron Blue F3G-A, the ligand of the packing material CB-10. The temperature was then shifted down to 20°C to change the structure of the stimulus-responsive polymer. It was confirmed that BSA was released from the packing material and eluted in the mobile phase due to the structural change of the polymer. The result is shown in Fig. 1.
  • the amount of BSA eluted in a temperature-dependent manner was 90% of the total amount of BSA adsorbed by CB-10.
  • the affinity-controlling material according to the present invention is advantageous in the following points.
  • the affinity-controlling material of the present invention When the affinity-controlling material of the present invention is used as an affinity chromatographic packing, the packing can be quickly regenerated compared with the conventional supports. 4)
  • the affinity-controlling material of the present invention makes it possible to separate and purify various types of target substances, which cannot be achieved by the conventional affinity chromatographic packings.

Abstract

Cette invention concerne un matériau à affinité contrôlée dans lequel un polymère sensible à un stimulus et un ligand présentant une affinité pour une substance cible sont liés indépendamment, de préférence par covalence, à une matrice de support. Ce matériau permet de séparer et de purifier une substance cible telle qu'une substance active au plan physiologique sous l'effet d'un stimulus physique tout en maintenant à une valeur constante au moins un paramètre autre que la température (par exemple pH d'une solution, concentration de solvants organiques ou concentration en sel) au moyen d'une matrice de support capable d'empêcher l'adsorption non spécifique de protéines et de garantir d'excellents résultats en termes de séparation.
EP00927741A 1999-05-11 2000-05-11 Materiau a affinite controlee avec polymere sensible a un stimulus et procede de separation/purification faisant intervenir ce materiau Withdrawn EP1194228A1 (fr)

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PCT/JP2000/003022 WO2000067901A1 (fr) 1999-05-11 2000-05-11 Materiau a affinite controlee avec polymere sensible a un stimulus et procede de separation/purification faisant intervenir ce materiau

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JP5109003B2 (ja) 2000-10-13 2012-12-26 岡野 光夫 刺激応答型アフィニティクロマトグラフィー材料等の分離材料および分離精製方法
JP4797143B2 (ja) * 2001-01-31 2011-10-19 独立行政法人産業技術総合研究所 触媒反応制御方法
JP4736362B2 (ja) * 2003-09-09 2011-07-27 学校法人東京電機大学 光および熱応答性吸着材料、可溶性物質の回収方法
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FR2902799B1 (fr) 2006-06-27 2012-10-26 Millipore Corp Procede et unite de preparation d'un echantillon pour l'analyse microbiologique d'un liquide
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