WO2016143215A1 - 吸着材、それを用いた分離精製装置及び分離精製方法 - Google Patents

吸着材、それを用いた分離精製装置及び分離精製方法 Download PDF

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WO2016143215A1
WO2016143215A1 PCT/JP2015/085071 JP2015085071W WO2016143215A1 WO 2016143215 A1 WO2016143215 A1 WO 2016143215A1 JP 2015085071 W JP2015085071 W JP 2015085071W WO 2016143215 A1 WO2016143215 A1 WO 2016143215A1
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temperature
adsorbent
target substance
site
responsive
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PCT/JP2015/085071
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English (en)
French (fr)
Japanese (ja)
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優史 丸山
靖彦 多田
啓介 渋谷
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株式会社日立製作所
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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
    • 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
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • 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
    • B01J20/34—Regenerating or reactivating
    • 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
    • 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
    • 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 an adsorbent, a separation and purification apparatus and a separation and purification method using the same.
  • affinity chromatography using an adsorbent having a specific affinity for the target substance is often used.
  • the target substance is selectively adsorbed on the adsorbent by bringing the liquid to be processed containing the desired target substance into contact therewith. Then, after removing impurities and the like contained in the liquid to be treated, the target substance is desorbed to separate and purify the target substance.
  • it is generally necessary to change the pH, salt concentration, etc. to reduce the affinity between the target substance and the adsorbent.
  • the target substance is desorbed by changing the pH to an acidic range or increasing the salt concentration, the target substance may be denatured and deteriorated. Further, after the separation and purification treatment, neutralization treatment of an acidic solution, dialysis treatment of a high salt concentration solution, and the like are required, and thus there are difficulties such as an increase in man-hours and waste and a reduction in recovery rate.
  • management operations that minimize the time for exposing biomolecules to a temperature condition of 42 ° C. or higher and an acidic condition of pH 5 or lower are desired because of quality control requirements. Yes. Therefore, as a means for desorbing the target substance adsorbed on the adsorbent, development of a method utilizing a phase change accompanying a temperature change of a temperature-responsive molecule has been advanced.
  • Patent Document 1 describes an adsorbent for cell separation having, on a substrate surface, a region in which a substance that specifically adsorbs target cells is introduced and a region to which a stimulus-responsive polymer is bonded.
  • a substance that specifically adsorbs target cells is introduced into the substrate via a polymer that has affinity for both the substance and the substrate, but not for cells. It is a thing. Specifically, it is described that poly (N-isopropylacrylamide) used as a temperature-responsive polymer was desorbed by lowering the temperature from 37 ° C. to 10 ° C. (See paragraphs 0061 and 0062).
  • Patent Document 2 describes an affinity control-type material in which an affinity substance (ligand) having affinity for a stimulus-responsive polymer and a target substance is independently bonded to a support, preferably covalently bonded. Yes. Specifically, it is described that the target substance was desorbed by lowering the temperature of poly (N-isopropylacrylamide) used as the stimulus-responsive polymer from 40 ° C. to 20 ° C. (paragraph 0061). reference).
  • Patent Document 3 includes a stimulus-responsive polymer and a substance that specifically interacts with a target substance, and the stimulus-responsive material polymer undergoes a structural change by physical stimulation, so that the target substance
  • a separation material containing a composite material in which the interaction of a substance that specifically interacts with the target is subjected to changes in the chemical or physical environment, and the interaction force with the target substance is reversibly changed by a physical stimulus.
  • a separation material characterized in that a stimulus-responsive polymer has no affinity for a substance that specifically interacts with a target substance is described. Specifically, it is described that the target substance was desorbed by lowering the poly (N-isopropylacrylamide) used as the stimulus-responsive polymer from 40 ° C. to 2 ° C. or 10 ° C. (See Example 2 and Example 3).
  • Patent Document 1 a separation and purification material (corresponding to an adsorbent) that desorbs a target substance by a phase change accompanying a temperature change of a temperature-responsive molecule is used.
  • these separation and purification materials desorb the target substance due to the phase change accompanying the temperature drop of the temperature-responsive molecule.
  • a biological material treated as a pharmaceutical or a pharmaceutical raw material is stored in a solution at a low temperature of about 4 ° C. so as not to impair the activity and molecular structure. Therefore, in order to desorb the target substance by the phase change accompanying the temperature decrease of the temperature-responsive molecule, it is necessary to perform a process in which the liquid to be processed is heated to the phase change temperature or higher prior to the separation and purification process.
  • the present invention is an adsorbent used for the separation and purification treatment of the target substance contained in the liquid to be treated, the adsorbent capable of appropriately performing the separation and purification treatment, a separation and purification apparatus using the same, and
  • An object is to provide a separation and purification method.
  • an adsorbent includes an adsorption site composed of a functional molecule that adsorbs a target substance in a detachable manner, a temperature response site composed of a temperature-responsive molecule that changes phase according to a temperature change, and An adsorption site and a carrier supporting the temperature response site, wherein the temperature response site has an action of desorbing a target substance adsorbed on the adsorption site by a phase change accompanying a temperature rise, and the adsorption site
  • the molecular weight of is characterized by being not more than twice the molecular weight of the temperature-responsive site.
  • the separation and purification apparatus is characterized by having the adsorbent and a column packed with the adsorbent.
  • the separation and purification method according to the present invention is a separation and purification method for separating and purifying a target substance contained in a liquid to be treated using a column filled with the adsorbent, wherein the adsorbent is An adsorption site composed of a functional molecule that adsorbs the target substance in a detachable manner, a temperature response site composed of a temperature-responsive molecule that changes phase according to a temperature change, and a carrier that immobilizes the adsorption site and the temperature response site.
  • the temperature-responsive part has an action of desorbing the target substance adsorbed on the adsorption part by a phase change accompanying a temperature rise, and the molecular weight of the adsorption part is less than twice the molecular weight of the temperature-responsive part
  • the separation and purification method is a separation and purification method for separating and purifying a target substance contained in a liquid to be treated using a column packed with the adsorbent, and the target substance is placed on the column.
  • Adsorption material which can perform a separation-purification process appropriately, Separation-purification apparatus using the same, and Separation and purification methods can be provided.
  • FIG. 1A shows a state before the target substance is adsorbed on the adsorbent
  • FIG. 1B shows a state where the target substance is adsorbed on the adsorbent
  • FIG. 1C shows a state where a phase change has occurred in the adsorbent
  • FIG. 1D shows a phase change. This shows a state where the target substance is desorbed from the adsorbent in which is generated.
  • FIG. 2A shows the step of binding the temperature-responsive molecule to the carrier
  • 2B shows the step of binding the functional molecule to the temperature-responsive molecule.
  • FIG. 1 is a diagram schematically showing the structure and operation of an adsorbent according to an embodiment of the present invention.
  • 1A shows a state before the target substance is adsorbed on the adsorbent
  • FIG. 1B shows a state where the target substance is adsorbed on the adsorbent
  • FIG. 1C shows a state where a phase change has occurred in the adsorbent
  • FIG. 1D shows a phase change. This shows a state where the target substance is desorbed from the adsorbent in which is generated.
  • the adsorbent 1 includes an adsorption site 10, a temperature response site 20, and a carrier 30.
  • the adsorbent 1 is a separation / purification material used for separation / purification of the target substance L contained in the liquid to be treated, and is, for example, a packing material for a separation / purification apparatus (corresponding to a column) for affinity chromatography. It is a suitable material.
  • the adsorbent 1 is applied with a solution containing the desired target substance L together with other impurities as a liquid to be treated for separation and purification (see FIG. 1A).
  • IgG is exemplified as the target substance L, but the target substance L is not limited to this.
  • the target substance L in the liquid to be treated is selectively adsorbed on the adsorption site 10 of the adsorbent 1 with a relatively high affinity.
  • the impurities contained in the liquid to be treated have a relatively low affinity for the adsorption site 10 and are not adsorbed or weakly adsorbed, so that the target substance L is left by the washing treatment. Removed.
  • the adsorbent 1 according to the present embodiment has a function of desorbing the target substance L due to a phase change accompanying a temperature rise of the temperature-responsive molecule (corresponding to the temperature-responsive portion 20). Therefore, when the temperature of the adsorbent 1 is increased, the temperature response portion 20 changes to a three-dimensional structure in an aggregated state as shown in FIG. 1C. As a result, the affinity between the adsorption site 10 and the target substance L decreases, and the target substance L adsorbed on the adsorption site 10 is desorbed (see FIG. 1D). In the adsorbent 1 according to the present embodiment, the separation and purification process of the desired target substance L contained in the liquid to be processed is thus realized.
  • the adsorbent 1 has a structure in which the branch-like structure schematically shown in FIG. 1 is repeated innumerably, and a large number of adsorption sites 10 and temperature response sites 20 are supported on the carrier 30. And such a support
  • carrier 30 comprises the isolation
  • the liquid to be treated that is treated in the separation and purification treatment using the adsorbent 1 is an aqueous solution containing the desired target substance L that specifically adsorbs to the adsorption site 10. That is, a solution containing a desired target substance L having a high affinity with the adsorption site 10 and impurities or the like having a relatively low affinity with the adsorption site 10 is provided for the treatment.
  • the aqueous solution may be either an aqueous solution using water as a solvent or a mixed solvent solution of water and an organic solvent. It should be noted that the solution herein includes a suspension or dispersion conceptually.
  • the adsorption site 10 is composed of a functional molecule that adsorbs the target substance L in a detachable manner.
  • the functional molecule In the low temperature range lower than the phase change temperature of the temperature response site 20, the functional molecule exhibits a strong affinity for the target material L and specifically adsorbs the target material L, and is higher than the phase change temperature of the temperature response site 20. In the high temperature range, the affinity is weakened so that the target substance L can be detached.
  • the adsorption of the target substance L to the adsorption site 10 may be either a chemical bond or a physical bond, or a multipoint bond.
  • a chemical bond for example, hydrogen bond, hydrophobic interaction, ⁇ - ⁇ interaction, dipole-dipole interaction, charge-dipole interaction, charge-charge interaction, charge transfer interaction, van der Waals bond, anchor effect, etc.
  • a reversible bond is formed by an appropriate interaction or a combination thereof.
  • an appropriate substance such as a biological substance, a cell, an organic compound, an inorganic compound, a complex, a metal ion, an inorganic fine particle, or a metal fine particle is applied as long as it is a substance that specifically binds to the adsorption site 10.
  • a substance that is desired to be stored in a low temperature range of about 0 ° C. or higher and 20 ° C. or lower, or a substance that has low stability in an acidic range of about pH 5 or lower is preferable.
  • target substance L examples include biological substances, cells, acid-reactive substances that react intramolecularly with acids having a pH of about 5 or less, or intermolecular reactions with impurities, etc., and decompose by acids with a pH of about 5 or less.
  • examples include acid-decomposable substances that are generated.
  • Examples of biological substances include proteins, peptides, amino acids, saccharides, nucleic acids, lipids, metabolites, and the like.
  • Examples of the cells include human cells, animal cells, plant cells, and microbial cells including bacteria and fungi. As cells, blood cells, recombinant cells, fused cells, and the like can be applied.
  • Examples of the acid-reactive substance and the acid-decomposable substance include various substances such as esters and amides.
  • any functional low molecule or functional polymer having an appropriate structure may be used as long as it is a molecule that specifically adsorbs the target substance to be separated and purified. it can.
  • functional small molecules include 5-membered or 6-membered aromatic or heteroaromatic rings, sulfide groups, sulfonyl groups, amide groups, amino acids, nucleic acids, sugars, and hydrophobicity such as cyclophane and calixarene.
  • a functional molecule having at least one selected skeleton it is easy to form an interaction by multipoint bonding, particularly when a protein, nucleic acid, or the like is used as a target substance.
  • the sulfide group is preferable in that it can form a functional molecule exhibiting antibody binding ability by thiophilic interaction.
  • a functional polymer it is preferable to use the functional molecule which consists of a peptide, protein, polysaccharide, polyacrylamide derivative, polymethacrylic acid derivative, polyfunctional polyamide, polyfunctional polyester, etc., for example.
  • a water-soluble stable functional polymer facilitates the molecular design of the adsorption site 10.
  • the combination of the functional molecule constituting the adsorption site 10 and the target substance L include antibodies, antigens, protein A, protein G, protein L, Fc receptors, peptide fragments thereof, or antibody adsorption capacity
  • a combination with a nucleic acid, a combination of biotin and avidin, or streptavidin can be exemplified.
  • the adsorption site 10 is supported by the carrier 30.
  • the adsorption site 10 is bonded to the side chain of the temperature response site 20, but it may be bonded to the end of the temperature response site 20, or only the carrier 30 instead of the temperature response site 20. It may be directly bonded, or may be directly bonded to both the temperature response site 20 and the carrier 30.
  • part 10 may be couple
  • the adsorption site 10 may be bonded to the temperature response site 20 or the carrier 30 via a linker (also referred to as a spacer).
  • the linker is preferably a molecule that does not irreversibly adsorb the target substance.
  • the bond between the adsorption site 10 and the temperature response site 20 or the carrier 30 may be either a covalent bond or a non-covalent bond.
  • the bond between the adsorption site 10 and the temperature responsive site 20 or the carrier 30 is a covalent bond by an appropriate reaction such as a nucleophilic addition reaction, a nucleophilic substitution reaction, a radical addition reaction, a rearrangement reaction, a sigmatropy reaction, a condensation reaction, Formed by non-covalent bonds such as multipoint adsorption, hydrogen bonding, hydrophobic interaction, ⁇ - ⁇ interaction, dipole-dipole interaction, charge-dipole interaction, charge-charge interaction, charge transfer interaction, etc. can do.
  • epoxide and nucleophile carboxylic acid or activated carboxylic acid and nucleophile, alkyl halide and nucleophile, aryl halide and nucleophile, unsaturated carbonyl and nucleophile, unsaturated Reactions such as nitrile and nucleophile, amine and aldehyde, silanol and silane coupling agent, and binding by biotin and avidin, biotin and streptavidin, etc.
  • carboxylic acid or activated carboxylic acid and nucleophile alkyl halide and nucleophile
  • aryl halide and nucleophile unsaturated carbonyl and nucleophile
  • unsaturated Reactions such as nitrile and nucleophile, amine and aldehyde, silanol and silane coupling agent, and binding by biotin and avidin, biotin and streptavidin, etc.
  • the molecular weight of the adsorption site 10 is not more than twice the molecular weight of the temperature response site 20 described later.
  • the molecular weight of the adsorption site 10 is made smaller than the molecular weight of the temperature response site 20 in this way, the recovery rate of the target substance L in the separation and purification process is increased, and a recovery rate of about 30% or more can be realized.
  • the ratio between the molecular weight of the adsorption site 10 and the temperature response site 20 can be changed to further improve the recovery rate.
  • the molecular weight of the adsorption site 10 is preferably 0.5 times or less than the molecular weight of the temperature response site 20, and a recovery rate of 70% or more is required.
  • the molecular weight of the adsorption site 10 is preferably 0.2 times or less than the molecular weight of the temperature response site 20.
  • the molecular weight of the adsorption site 10 is preferably 40 or more. When the molecular weight of the adsorption site 10 is 40 or more, a predetermined target substance can be specifically adsorbed.
  • the temperature responsive portion 20 is made of a temperature responsive molecule that changes phase with temperature change.
  • the temperature-responsive molecule indicates a lower critical solution temperature (Lower Critical Solution Temperature; LCST). That is, the temperature-responsive molecule is present in a solution to be treated in a low temperature region lower than the lower critical solution temperature, and is present in a phase-insoluble state in a high temperature region higher than the lower critical solution temperature. Therefore, the temperature-responsive part 20 changes phase with the lower critical solution temperature as a boundary and causes a remarkable change in the three-dimensional structure, thereby increasing the affinity between the adsorption part 10 and the target substance L bound to the adsorption part 10. Make it variable.
  • LCST Lower Critical Solution Temperature
  • the temperature response part 20 has an action of desorbing the target substance L adsorbed on the adsorption part 10 by a phase change accompanying a temperature rise.
  • the temperature response part 20 is heated from a low temperature range lower than the lower critical solution temperature (corresponding to the phase change temperature) indicated by the temperature responsive molecule to a higher temperature range higher than the lower critical solution temperature, the phase change occurs.
  • the affinity between the adsorption site 10 and the target substance L adsorbed on the adsorption site 10 is lower than that at the time of adsorption, and the target substance L is desorbed from the adsorbent 1. .
  • Temperature-responsive polymers are acrylamide derivatives, methacrylic acid derivatives such as methacrylic acid esters, acrylic acid derivatives such as acrylic acid esters, vinyl alcohol derivatives, N-modified ⁇ -polylysine derivatives as monomers (monomer units).
  • ⁇ -polylysine derivatives such as ⁇ -glutamic acid amide (corresponding to glutamic acid derivatives), ⁇ -aspartic acid derivatives such as ⁇ -aspartic acid amide derivatives (aspartic acid) Corresponding to derivatives)), lactic acid, diol derivatives, caprolactams, caprolactones, sugars, siloxanes, and the like.
  • the temperature-responsive molecule may be a polymer of any of these monomers or a copolymer of a combination of these monomers.
  • the copolymer may be any of random type, block type and graft type.
  • These monomers are independently of each other a hydroxy group, carboxyl group, amino group, thiol group, epoxy group, nitrile group, halogen atom, methyl group, ethyl group, n-propyl group, iso-propyl group, n Alkyl groups such as -butyl group, sec-butyl group, iso-butyl group, tert-butyl group, n-pentyl group, n-hexyl group, etc., cycloalkyl groups optionally having these as substituents, alkoxy It may be substituted with a substituent (functional group) such as a group, an aryl group, an aralkyl group, an acyl group, and an aminoacyl group.
  • the temperature-responsive polymer is not limited to a linear polymer, and may be a branched polymer, a dendrimer, a gel, a crosslinked gel, or the like.
  • Examples of the acrylamide derivatives include N-isopropylacrylamide, N-diisopropylacrylamide, N-ethylacrylamide, N-diethylacrylamide, N-methylacrylamide, N-dimethylacrylamide, and other N-substituted derivatives.
  • Examples of the methacrylic acid derivative include butyl methacrylate, propyl methacrylate, ethyl methacrylate, methyl methacrylate, and other O-substituted derivatives.
  • Examples of the acrylic acid derivative include butyl acrylate, propyl acrylate, ethyl acrylate, methyl acrylate, and other O-substituted derivatives.
  • Examples of the vinyl alcohol derivative include vinyl alcohol methyl ether, vinyl alcohol acetylate, and other O-substituted derivatives.
  • Examples of the ⁇ -polylysine derivative include lysine valeric acid amide, lysine butyric acid amide, lysine propionic acid amide, N-hydroxypentyllysine, N-hydroxybutyllysine, N-hydroxypropyllysine, and other N-substituted derivatives. Can be mentioned.
  • Examples of the ⁇ -glutamic acid derivative include glutamic acid hydroxyhexylamide, glutamic acid hydroxypentylamide, glutamic acid hydroxybutylamide, glutamic acid hydroxypropylamide, and other O-substituted derivatives.
  • Examples of the ⁇ -aspartic acid derivative include aspartic acid hydroxyhexylamide, aspartic acid hydroxypentylamide, aspartic acid hydroxybutylamide, aspartic acid hydroxypropylamide, and other O-substituted derivatives.
  • Examples of the diol derivative include polyethylene glycol and polypropylene glycol.
  • Examples of the saccharide include hydroxypropyl cellulose.
  • a temperature-responsive low molecule that exhibits a lower critical solution temperature can be used together with a temperature-responsive polymer that exhibits a lower critical solution temperature.
  • a temperature-responsive low molecule for example, spiropyran, azobenzene, diarylethene, derivatives thereof, liquid crystal molecules, and the like that exhibit temperature responsiveness as well as photoresponsiveness can be used.
  • the phase change temperature of the temperature response part 20 is preferably in the range of 0 ° C. or more and 60 ° C. or less, more preferably in the range of 0 ° C. or more and 40 ° C. or less, and in the range of 10 ° C. or more and 40 ° C. or less. Is more preferable.
  • the phase change temperature of the temperature responsive part 20 is in such a temperature range, the target substance L can be adsorbed and desorbed under mild temperature conditions, so that deterioration of the target substance L can be avoided. It is.
  • the phase change temperature of the temperature responsive part 20 is affected by the solvent, impurities, pH, etc.
  • the molecular design is such that the temperature responsive part 20 changes phase under pH conditions of pH 6-8. Is preferred.
  • Such molecular design of the temperature responsive portion 20 can be performed by introducing various functional groups into the temperature responsive molecule. For example, the balance between hydrophilic group and hydrophobic group, introduction of charge interaction, etc. Can be realized.
  • the temperature response part 20 is supported by the carrier 30.
  • the temperature responsive portion 20 is bonded to the carrier 30 via the side chain, but may be bonded to the carrier 30 via the end.
  • a single temperature response site 20 may be bonded to a single carrier 30, or a plurality of temperature response sites 20 may be bonded.
  • the temperature responsive portion 20 may be bonded to the carrier 30 through a linker.
  • the bond between the temperature response site 20 and the carrier 30 may be either a covalent bond or a non-covalent bond.
  • the bond between the temperature response site 20 and the carrier 30 can be formed as described above in the same manner as the bond between the adsorption site 10 and the temperature response site 20 or the carrier 30.
  • the carrier 30 supports the adsorption site 10 and the temperature response site 20.
  • the carrier 30 preferably has high chemical and physical stability in the liquid to be treated, and preferably gives the adsorbent 1 mechanical strength, shapeability, handleability, and the like.
  • the carrier 30 can have an appropriate shape, and may be porous or non-porous. Specific examples of the shape of the carrier 30 include a plate shape, a bead shape, a fiber shape such as a nonwoven fabric and a woven fabric, a film shape, a monolith shape, and a hollow fiber shape.
  • the material of the carrier 30 may be either an organic material or an inorganic material, or a composite material using these materials in combination.
  • polysaccharides such as agarose, sepharose, cellulose, polystyrene, polyalkyl methacrylate, polyglycidyl methacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polysiloxane, polyfluorinated ethylene, polyethylene, polypropylene, polyethylene terephthalate.
  • Synthetic resins such as polytrimethylene terephthalate, polyvinyl chloride, polyvinyl acetate, polycarbonate, PA6, PA66, PA11, PA12, carbon materials such as graphite, carbon fiber, carbon nanotube, fullerene, silica, alumina, titania, Oxides such as zirconia and iron oxide, carbonates such as calcium carbonate, phosphates such as hydroxyapatite and aluminum phosphate, borosilicate glass, etc.
  • Silicates such as oxalate, silicate, diatomaceous earth, sulfates such as aluminum sulfate and calcium sulfate, inorganic compounds such as silicon carbide and silicon nitride, ferrite, permalloy, chromium steel, iron-aluminum alloy, gold, silver, Metal materials such as platinum, palladium, and rhodium can be used.
  • preferred materials are agarose, sepharose, cellulose, polystyrene, silica, iron oxide or ferrite useful as a magnetic carrier from the viewpoints of stability and ease of production.
  • the carrier 30 may have an appropriate reactive group such as a carboxy group, an amino group, or a hydroxy group that forms a covalent bond with the adsorption site 10 or the temperature response site 20 on the surface.
  • the carrier 30 may be surface-modified with molecules other than the adsorption site 10 and the temperature response site 20. For example, blocking that prevents non-specific adsorption of the target substance, surface modification that controls the orientation of the adsorption site 10 or the temperature response site 20, surface modification that modifies the dispersibility or adsorption of the carrier 30, etc. It may be done.
  • the adsorbent 1 according to the present embodiment can be manufactured using a conventionally known appropriate reaction.
  • the functional molecule constituting the adsorption site 10, the temperature responsive molecule constituting the temperature responsive site 20, the carrier 30 having a reactive group, etc. can be prepared using a known polymer reaction or the like, It can be obtained by using an activated surface treatment technique or by utilizing a separation / purification technique for natural products and biochemical products.
  • Each bond of the functional molecule constituting the adsorption site 10, the temperature responsive molecule constituting the temperature response site 20, and the carrier 30 having a reactive group is, for example, the functional molecule constituting the adsorption site 10 and the temperature response.
  • a method of binding the temperature-responsive molecule constituting the site 20 and then binding to the carrier 30; and the functionality of constituting the adsorption site 10 after binding the temperature-responsive molecule constituting the temperature-responsive site 20 to the carrier 30 A method of binding a molecule to a temperature-responsive molecule constituting the temperature-responsive site 20, and a method of binding the functional molecule constituting the adsorption site 10 and the temperature-responsive molecule constituting the temperature-responsive site 20 together with the carrier 30 Etc. can be taken.
  • FIG. 2 is a diagram showing an example of a method for manufacturing an adsorbent according to an embodiment of the present invention.
  • FIG. 2A shows the step of binding the temperature-responsive molecule to the carrier
  • FIG. 2B shows the step of binding the functional molecule to the temperature-responsive molecule.
  • the temperature responsive molecule constituting the temperature responsive portion 20 is bonded to the carrier 30, and then the functional molecule constituting the adsorbed portion 10 is constituted in the temperature responsive portion 20.
  • the method of binding to a temperature-responsive molecule is illustrated. In this method, a carrier having a carboxy terminus is used as the carrier 30, and glutaraldehyde is used as a linker between the adsorption site 10 and the temperature response site 20.
  • the carrier 30 is activated by reacting the carboxy terminus of the carrier 30 with carbodiimide hydrochloride (EDC ⁇ HCl). This reaction may be performed, for example, at pH 5.8 and 37 ° C. for about 2 hours. Subsequently, after removing unreacted substances and by-products, the activated carrier 30 is reacted with the temperature response site 20. This reaction may be performed, for example, at pH 5.8 and 37 ° C. for about 2 hours. Through such a reaction, the temperature-responsive molecule 20 and the carrier 30 can be bound via the nucleophilic amino group of the temperature-responsive site 20 (see the right figure in FIG. 2A).
  • EDC ⁇ HCl carbodiimide hydrochloride
  • the temperature-responsive molecule is bound to the carrier 30 under mild conditions such as a temperature condition of about 4 ° C. to 40 ° C. and a pH condition of about pH 4 to about 7 or less. It is also possible to make it.
  • the amino group of lysine (Lys) at the temperature-responsive site 20 is reacted with glutaraldehyde and activated.
  • This reaction may be performed, for example, at pH 9.0 and 37 ° C. for about 2 hours.
  • unreacted substances are removed, and then glutaraldehyde bound to the temperature response site 20 is reacted with the amino group of lysine (Lys) at the adsorption site 10.
  • This reaction may be performed at, for example, pH 7.4 and 37 ° C. for about 2 hours.
  • the temperature-responsive molecule 20 and the adsorption site 10 can be bound via glutaraldehyde.
  • the reaction can be allowed to proceed even when the temperature condition is from room temperature to about 37 ° C., the pH condition is in the range of about pH 6 to 9, and even more neutral.
  • the adsorbent 1 can be produced under mild conditions of temperature and pH, so that denaturation of the temperature-responsive molecule 20 and the adsorption site 10 can be avoided, and the adsorbent 1 Performance can be easily secured, and generation of acid and alkali waste liquids can be reduced.
  • FIG. 2 cross-linking by glutaraldehyde is schematically shown. However, since cross-linking by multimers of glutaraldehyde can be formed in an actual reaction, steric hindrance and interaction between molecules are partially eliminated. As a result, it may be easy to ensure a certain level of performance for the adsorbent.
  • FIG. 3 is a diagram illustrating an example of a schematic configuration of a manufacturing system including a separation and purification apparatus to which the adsorbent according to the embodiment of the present invention is applied.
  • the adsorbent 1 can be applied as a column packing material of a separation and purification apparatus 70 for affinity chromatography provided in the manufacturing system 100.
  • the manufacturing system 100 is a device that manufactures biological materials that are used as pharmaceuticals, pharmaceutical raw materials, and the like, and includes a culture tank 50, a storage tank 60, and a separation and purification apparatus 70. Further, the separation and purification device 70 is provided with a heating means 80.
  • the separation and purification apparatus 70 to which the adsorbent 1 according to the present embodiment is applied plays a role of separating and purifying biomaterials produced biochemically by culture from impurities in such a manufacturing system 100.
  • the separation and purification device 70 has the adsorbent 1 and a column packed with the adsorbent 1 in detail.
  • the column is composed of a hollow container having an arbitrary shape, and the column is filled with a liquid to be treated containing the target substance L, a washing liquid for washing impurities, an eluent for desorbing the target substance L, and the like.
  • a liquid inlet and a liquid outlet for passing through the adsorbent 1 are provided.
  • the separation and purification device 70 is incorporated in the manufacturing system 100 by connecting pipes to the liquid inlet and the liquid outlet.
  • the material of the column of the separation and purification device 70 may be either an organic material or an inorganic material, or a composite material using these materials in combination. Specifically, for example, glass, stainless steel, aluminum alloy, polyethylene, polypropylene, polyethylene terephthalate, polytrimethylene terephthalate, polyvinyl chloride, polyvinyl acetate, polycarbonate, PA6, PA66, PA11, PA12 and the like can be used. .
  • a heat insulating material is interposed in the column.
  • the heat insulating material can be installed, for example, between the packed adsorbent 1 and the column inner wall or around the column outer wall. By adiabatic between the filler 1 and the outside air, heat loss due to the temperature rise of the adsorbent 1 is reduced, and an efficient temperature rise is enabled by heat conduction in the filler 1. Cost and heating time can be suppressed more effectively.
  • a foam type heat insulating material such as urethane foam or phenol foam
  • a fiber type heat insulating material such as glass fiber, mineral fiber, or resin fiber, a vacuum heat insulating material, or a combination thereof may be used. it can.
  • the heating means 80 has a function of increasing the temperature of the adsorbent 1 provided in the separation and purification apparatus 70 with the phase change temperature of the temperature-responsive molecule interposed therebetween. The temperature of the adsorbent is raised by the heating means 80 beyond the phase change temperature, so that the target substance L adsorbed on the adsorbent 1 is desorbed.
  • the heating means 80 preferably has a function of adjusting the temperature of the adsorbent 1 in a range of at least 0 ° C. and 40 ° C., preferably in a range of 0 ° C. and 60 ° C.
  • the heating means 80 is attached to the column of the separation and purification apparatus 70 and is configured to heat the adsorbent 1 packed in the column inside the column. It is also possible to install it independently and contact the adsorbent 1 on which the target substance L is adsorbed after heating the desorption liquid (also called eluate) for desorbing the target substance outside the column. Good. Further, when the heating means 80 is attached to the column, even if it is a type in which heat is transferred from the outside of the column, a heat transfer tube extending inside the column is provided to control the temperature inside the column. The format to be performed may be used.
  • the temperature is adjusted in an in-line configuration in which the temperature is adjusted in the pipe through which the desorbed liquid flows, and in the storage tank in which the desorbed liquid is temporarily stored.
  • predetermined cells are cultured in the culture tank 50, and a desired biological material is produced biochemically by the cells.
  • a desired biological material is produced biochemically by the cells.
  • antibody production or the like is performed by culturing genetically modified animal cells or the like into which an antibody gene has been introduced.
  • the produced solution containing the biological material is transferred to the storage tank 60 and temporarily stored under a low temperature condition of about 4 ° C., for example.
  • the solution containing the biological material is transferred to the separation and purification device 70, and the separation and purification device 70 performs separation and purification processing using the biological material as the target material L.
  • the separation and purification method according to the present embodiment uses the column (corresponding to the separation and purification apparatus 70) packed with the adsorbent 1 to separate and purify the target substance L contained in the liquid to be treated. It is a method to do.
  • This separation and purification method is a method that includes an adsorption step, a washing step, a desorption step, and an elution step sequentially.
  • the liquid to be treated containing the target substance L is passed through the column filled with the adsorbent 1, and the target substance is adsorbed to the adsorption site 10 provided in the adsorbent 1.
  • the liquid to be treated include a culture liquid obtained in the culture tank 50, a filtrate obtained by filtering the culture liquid, and a low-temperature solution.
  • the temperature of the liquid to be treated is preferably 0 ° C. or higher and not higher than the phase change temperature of the temperature-responsive molecule, more specifically, preferably 0 ° C. or higher and 20 ° C. or lower, more preferably The range is from 0 ° C. to 15 ° C., more preferably from 0 ° C.
  • a more preferable form of the separation and purification method according to the present embodiment is a method including a storage step of storing the liquid to be processed containing the target substance L in such a low temperature region as a pre-step of the adsorption step.
  • the adsorption process is performed with the temperature condition being lower than the phase change temperature of the adsorbent 1.
  • the adsorption temperature is preferably in the range of 0 ° C. to 20 ° C., more preferably in the range of 0 ° C. to 15 ° C., and in the range of 0 ° C. to 10 ° C. Further preferred.
  • the target substance is adsorbed at such a temperature
  • the target substance L can be adsorbed by allowing the liquid to be treated to pass through the column without adjusting the temperature in advance. Can be avoided.
  • about other conditions, such as pH conditions and salt concentration you may make liquid flow constant.
  • the column through which the liquid to be treated is passed is preferably preliminarily adjusted so that the internal temperature of the column is lower than the phase change temperature of the adsorbent 1.
  • the adsorbent 1 on which the target substance L is adsorbed is washed under the condition that the target substance L is not desorbed.
  • impurities or the like that are not adsorbed or weakly adsorbed due to low affinity for the adsorption site 10 and the unadsorbed target substance L are removed.
  • the washing step is performed with the temperature condition lower than the phase change temperature of the adsorbent 1 so that the target substance L is not desorbed by washing.
  • the cleaning temperature is preferably in the range of 0 ° C. to 20 ° C., more preferably in the range of 0 ° C. to 15 ° C., and in the range of 0 ° C. to 10 ° C. Further preferred. Other conditions such as pH and salt concentration may be constant.
  • an appropriate buffer solution such as PBS (Phosphate Buffered Saline) can be used as PBS (Phosphate Buffered Saline) can be used.
  • the adsorbent 1 preferably has a phase change temperature of the temperature response portion 20 in the range of 0 ° C. or higher and 40 ° C. or lower, and more preferably in the vicinity of 20 ° C. (meaning 10 ° C. or higher and 30 ° C. or lower). preferable.
  • the desorption step is performed by raising the temperature condition to a temperature higher than the phase change temperature of the adsorbent 1.
  • the desorption temperature is preferably in the range of 21 ° C. to 40 ° C., more preferably in the range of 25 ° C. to 40 ° C., and in the range of 30 ° C. to 40 ° C. Is more preferable. If it is such temperature, the heating cost required for temperature rising of the adsorbent 1 will not become excessive, and thermal denaturation of the target substance L can also be avoided.
  • the temperature of the adsorbent 1 can be increased by passing the heated desorption liquid or by heating the adsorbent 1 and the cleaning liquid passed through the adsorbent 1.
  • an appropriate buffer solution such as PBS can be used.
  • the desorbed target substance L is eluted from the column.
  • the target substance L desorbed from the adsorption site 10 is eluted and recovered from the column, and the separation and purification process is completed.
  • the elution step may be performed integrally with the desorption step by passing the desorption solution and then collecting it at the liquid outlet of the column.
  • the elution step can be performed at the same temperature as the desorption temperature or at a temperature lower than the desorption temperature. From the viewpoint of reducing the deterioration of the target substance, it is preferable to pass an eluate having a low temperature of about 10 ° C. or lower.
  • an appropriate buffer such as PBS can be used as the eluate.
  • the desorption of the target substance L adsorbed on the adsorbent 1 is heated or adsorbed on the desorbed liquid that is passed through the column. This can be achieved by either heating the column filled with the material 1 and the cleaning liquid passed through the adsorbent 1, and the total heat capacity of the object to be heated is relatively low.
  • FIG. 4 is a diagram illustrating an example of a schematic configuration of a manufacturing system including a separation and purification apparatus to which an adsorbent according to a comparative example is applied.
  • a conventional adsorbent that desorbs a target substance by a phase change accompanying a temperature drop of a temperature-responsive molecule (also referred to as an adsorbent according to a comparative example) is applied as a column packing material for affinity chromatography, the temperature responsiveness The target substance L is adsorbed in a high temperature range higher than the phase change temperature of the molecule, and after the washing process, the temperature is lowered to a low temperature range lower than the phase change temperature to desorb the adsorbed target substance L. There must be. Therefore, in the manufacturing system 100C including the separation and purification device 70C to which the adsorbent according to the comparative example is applied, as shown in FIG.
  • the pipe on the liquid inlet side (upstream side) of the separation and purification device 70C is provided with heating means 90C.
  • a process of raising the temperature of the liquid to be treated containing the biological material to the phase change temperature or higher is required.
  • the manufacturing system 100C After culturing in the culture tank 50, it is necessary to preheat the whole of the large-volume culture solution stored in the storage tank 60 under low temperature conditions. It takes time.
  • the separation and purification apparatus and the separation and purification method to which the adsorbent 1 according to the present embodiment is applied the target substance L adsorbed on the adsorbent 1 is changed in phase with the temperature rise of the temperature response portion 20. Therefore, it is not necessary to raise the temperature of a large volume of liquid to be processed in advance.
  • the column contents (adsorbent 1 and adsorbent 1 having a smaller total heat capacity than the culture solution or the like, or a desorbed solution that is smaller than the culture solution or the like.
  • the target substance L can be desorbed by heating in any one of (2). Therefore, the heating cost and the heating time are suppressed, the deterioration of the target substance in the process is reduced, the process efficiency is hardly impaired, and the separation and purification process can be appropriately performed. Furthermore, the effect of improving the treatment efficiency can be made more advantageous as the volume of the liquid to be treated and the amount of adsorbent adsorbed are larger, and as the concentration of the target substance contained in the liquid to be treated is lower.
  • the adsorbent according to Examples 1 to 3 is manufactured, and the correlation of the molecular weight ratio between the adsorption site 10 and the temperature response site 20 with respect to the recovery rate of the target substance L is evaluated. Went.
  • the adsorbents according to Comparative Examples 1 to 3 were produced and evaluated in the same manner as a control for the adsorbents according to the examples of the present invention.
  • an ⁇ -polylysine derivative represented by the following chemical formula was used as the temperature responsive portion 20.
  • X is an n-butoxycarbonyl group (-nBuCO)
  • m is an integer of 1 or more
  • n is an integer greater than m
  • the copolymerization format is arbitrary.
  • the lower critical solution temperature of this ⁇ -polylysine derivative is 28 ° C.
  • the carrier 30 a polystyrene microplate having a carboxy terminus was used as the carrier 30, as the carrier 30, a polystyrene microplate having a carboxy terminus was used.
  • Example 1 As the adsorbent according to Example 1, an adsorbent in which the adsorption site 10 is (3-thia-5-pyridylpentyl) sulfonyl represented by the following chemical formula was produced as follows. This adsorption site is a functional molecule having antibody adsorption ability by thiophilic interaction.
  • EPL-V80 The obtained ⁇ -polylysine derivative (EPL-V80) was reacted with 1 equivalent of (3-thia-5-pyridylpentyl) vinylsulfone, so that 80% of the side chain was converted to valeric acid amide.
  • An ⁇ -polylysine derivative (EPL-V80-S5) was obtained in which 5% of the chain was converted to (3-thia-5-pyridylpentyl) sulfonylethylamine.
  • Example 2 As an adsorbent according to Example 2, amino (4-hydroxyphenylethylamino) (phenylamino) triazine ((4- [2- (4-hydroxyphenyl) ethylamino] -6) having an adsorption site 10 represented by the following chemical formula: An adsorbent which is -phenylamino-1,3,5-triazin-2-yl) amine) was prepared as follows.
  • the adsorption site 10 is a functional molecule having an antibody adsorption ability that mimics protein A.
  • ⁇ -polylysine derivative (EPL-V57) was reacted with 1 equivalent of chloro (4-hydroxyphenylethylamino) (phenylamino) triazine, and 57% of the side chain was converted to valeric acid amide.
  • an ⁇ -polylysine derivative (EPL-V57-T10) in which 10% of the side chain was converted to amino (4-hydroxyphenylethylamino) (phenylamino) triazine was obtained.
  • Example 3 As the adsorbent according to Example 3, an adsorbent in which the adsorption site 10 is an extracellular domain having protein A antibody binding ability was produced as follows.
  • EPL-V80 ⁇ -polylysine derivative in which 80% of the side chains were converted to valeric acid amide was obtained.
  • Protein A was cleaved with endoprotease “Glu-C”, and the resulting digest was fractionated by gel chromatography to recover a protein A fragment corresponding to a molecular weight of about 5000.
  • the carrier 30 to which the temperature response site 20 was bound was activated with glutaraldehyde, and the collected protein A fragment PBS solution (pH 7.4) was reacted to obtain the adsorbent according to Example 3.
  • EPL-V80 ⁇ -polylysine derivative in which 80% of the side chains were converted to valeric acid amide was obtained.
  • Protein A was cleaved with endoprotease “Glu-C”, and the resulting digest was fractionated by gel chromatography to recover a protein A fragment corresponding to a molecular weight of about 24,000.
  • the adsorbent according to Comparative Example 1 was prepared by activating the carrier 30 to which the temperature response site 20 was bound with glutaraldehyde and reacting the recovered protein A fragment with PBS solution (pH 7.4).
  • the adsorbent according to Comparative Example 2 was obtained by activating the carrier 30 to which the temperature response site 20 was bound with glutaraldehyde and reacting with a PBS solution of protein A (pH 5.8).
  • a biotinylated IgG PBS solution pH 7.4 was added to 6 wells of each adsorbent carrier 30 (polystyrene microplate) and allowed to stand at 4 ° C. for 30 minutes. Biotinylated IgG was adsorbed.
  • each well of the adsorbent carrier 30 (polystyrene microplate) was washed with a PBS solution (pH 7.4) to remove unadsorbed biotinylated IgG.
  • the three wells were heated from 4 ° C. to 37 ° C. and then allowed to stand for 1 hour to desorb the biotinylated IgG that had been adsorbed. During this time, the remaining three wells were stored at 4 ° C. without being heated.
  • FIG. 5 is a diagram showing the correlation of the molecular weight ratio between the adsorption site and the temperature response site with respect to the recovery rate of the target substance.
  • the vertical axis represents the recovery rate of biotinylated IgG (elution amount / total adsorption amount), and the horizontal axis represents the molecular weight ratio between the adsorption site and the temperature response site (molecular weight of the adsorption site / temperature response site).
  • the ⁇ plots are the values for the adsorbents according to Examples 1 to 3
  • the ⁇ plots are the values for the adsorbents according to Comparative Examples 1 to 2
  • the ⁇ plots are the temperature-responsive molecules.
  • the value (refer patent document 3: international publication 2001/74482) in the conventional adsorbent (as a control section) which desorbs a target substance using the phase change accompanying temperature fall is shown.
  • an ⁇ -polylysine derivative (EPL-V57-T10) obtained in the same manner as in Example 2 was reacted with a surface-activated bead carrier so that the adsorption site 10 was amino (4-hydroxyphenylethylamino) (phenyl An adsorbent was obtained which was amino) triazine, the temperature response site 20 was ⁇ -polylysine, and the carrier 30 was a surface activated bead carrier.
  • the PBS solution (pH 7.4) of the obtained adsorbent was filled in a hollow cylindrical column having a diameter of 10 cm so as to have a height of 20 cm, and a heat insulating material was wound on the peripheral wall of the column.
  • the liquid inlet and the liquid outlet of the column were respectively connected to a liquid feed pump and a heating device by pipes to obtain a separation and purification device according to the example.
  • the recovery rate by elution of the target substance L was about 80%.
  • 10 L of culture solution was used as the solution to be treated, whereas desorption of the target substance L adsorbed on the adsorption site 10 of the adsorbent was about 4 L of solution (PBS solution).
  • PBS solution 4 L of solution
  • Adsorbent 1 Adsorption site 20
  • Temperature response site 30 Carrier 50
  • Culture tank 60 Storage tank 70 Separation and purification device 80 Heating means 100 Manufacturing system

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WO2018096881A1 (ja) * 2016-11-25 2018-05-31 株式会社日立製作所 細胞培養培地、並びにこれを用いた細胞培養装置及び細胞培養方法
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CN110684150B (zh) * 2019-09-06 2021-05-11 南方医科大学 氨基酸纳米水凝胶及其制备方法和应用

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