WO2012121409A1 - Procédé de purification d'une substance physiologiquement active au moyen d'un support de chromatographie sensible à la température - Google Patents

Procédé de purification d'une substance physiologiquement active au moyen d'un support de chromatographie sensible à la température Download PDF

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Publication number
WO2012121409A1
WO2012121409A1 PCT/JP2012/056319 JP2012056319W WO2012121409A1 WO 2012121409 A1 WO2012121409 A1 WO 2012121409A1 JP 2012056319 W JP2012056319 W JP 2012056319W WO 2012121409 A1 WO2012121409 A1 WO 2012121409A1
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temperature
active substance
physiologically active
responsive
chromatography carrier
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PCT/JP2012/056319
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English (en)
Japanese (ja)
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一郎 小熊
和雄 奥山
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旭化成メディカル株式会社
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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
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3861—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using an external stimulus
    • B01D15/3876—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using an external stimulus modifying the temperature
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns

Definitions

  • the present invention relates to a method for purifying a physiologically active substance using a temperature-responsive ion exchange chromatography carrier that shows a change in the binding property to the physiologically active substance with a change in temperature.
  • Immunoglobulin is a physiologically active substance that controls the immune reaction.
  • the antibody is obtained from blood of the immunized animal or a cell culture solution of cells possessing antibody-producing ability or an ascites culture solution of the animal.
  • blood and culture fluids containing these antibodies include proteins other than antibodies, or complex contaminants derived from the raw material used for cell culture, and in order to separate and purify antibodies from those impurity components A complicated and time-consuming operation is usually necessary.
  • Liquid chromatography is important for antibody separation and purification.
  • Chromatographic techniques for separating antibodies include gel filtration chromatography, affinity chromatography, ion exchange chromatography, reverse phase chromatography, and the like, and antibodies are separated and purified by combining these techniques.
  • Patent Document 1 discloses a filler containing a copolymer having a charge capable of changing the effective charge density on the surface of the stationary phase by temperature change, a method for producing the filler, and temperature responsiveness using the filler.
  • a chromatographic method is disclosed.
  • Patent Document 2 discloses a temperature-responsive chromatographic carrier in which a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized at a high density on the surface of a substrate by an atom transfer radical polymerization method. Yes.
  • Patent Document 3 discloses a temperature-responsive chromatographic support for growing and reacting a polymer having a charge and changing hydration power within a temperature range of 0 to 80 ° C. by an atom transfer radical method using isopropyl alcohol as a solvent. A manufacturing method is disclosed.
  • Patent Document 4 discloses that a high molecular weight physiologically active substance useful in the fields of biology, medicine, pharmacy and the like can be separated under a specific condition including an aqueous mobile phase within a temperature range of 0 to 80 ° C.
  • a method for producing a liquid chromatography carrier in which a solid surface is coated with a charged polymer that varies in sum is disclosed.
  • Non-Patent Document 1 discloses a temperature-responsive chromatography carrier having a carboxyl group prepared by an atom transfer radical polymerization method and a method for producing the same. Also disclosed is a monomer composition optimized for lysozyme separation in the monomer composition used in the atom transfer radical polymerization method.
  • Patent Document 5 discloses a separation method using mutant protein A (temperature-responsive protein A) whose binding property to an antibody changes due to a change in a three-dimensional structure accompanying a temperature change.
  • the conditions do not cause problems such as damage to biological components to be purified, loss of function, or generation of unintended functions. ), A physiologically active substance is purified.
  • the temperature-responsive ion exchange chromatography carrier has a characteristic that it can bind to an antibody at a high temperature and can elute the antibody at a temperature lower than the temperature at the time of binding.
  • the antibody solution is previously heated to a temperature equivalent to the temperature at which the temperature-responsive ion exchange chromatography carrier has binding properties to the antibody.
  • antibody purification is carried out on an industrial scale, in order to quickly raise the antibody solution to a desired temperature, it is necessary to heat-exchange the antibody solution with a heat medium that is as hot as possible.
  • an object of the present invention is to provide a method capable of efficiently purifying an antibody by temperature-responsive ion exchange chromatography without denaturing the antibody.
  • the present inventors have studied and developed from various angles.
  • the antibody before purifying the antibody with the temperature-responsive ion exchange chromatography carrier, the antibody is bound to the temperature-responsive affinity chromatography carrier in the low temperature region, and the temperature-responsive affinity chromatography carrier binds to the antibody.
  • the antibody is eluted from the temperature-responsive affinity chromatography carrier with a buffer solution in a high-temperature region where the temperature-responsive ion exchange chromatography carrier is a temperature region in which the temperature-responsive ion exchange chromatography carrier is binding to the antibody.
  • the present inventors have found that the antibody can be efficiently purified by suppressing the denaturation of the antibody by purifying the antibody with a cationic ion exchange chromatography carrier.
  • the technology shown in the present invention was completely unpredictable from the prior art, and is expected to develop into a novel antibody separation system that was never found in the prior art.
  • the present invention has been completed based on such findings.
  • the production method described in the present invention makes it possible to suppress antibody denaturation and efficiently purify the antibody using a temperature-responsive ion exchange chromatography carrier.
  • useful physiologically active substances such as antibodies can be separated and purified on an industrial scale by temperature change.
  • the present embodiment exemplify apparatuses and methods for embodying the technical idea of the present invention, and the technical idea of the present invention specifies the arrangement of components and the like as follows. Not what you want.
  • the technical idea of the present invention can be variously modified within the scope of the claims.
  • the method for purifying a physiologically active substance uses at least first and second temperature-responsive chromatography carriers.
  • the first temperature-responsive chromatography carrier has a property of adsorbing a physiologically active substance in a low temperature region and desorbing the physiologically active substance in a high temperature region.
  • a temperature-responsive affinity chromatography carrier can be used, and preferably a temperature-responsive protein A carrier can be used.
  • the second temperature-responsive chromatography carrier has the property of adsorbing the physiologically active substance in the high temperature region and desorbing the physiologically active substance in the low temperature region.
  • a temperature-responsive ion exchange chromatography carrier can be used as the second temperature-responsive chromatography carrier.
  • the low temperature region in the first temperature-responsive chromatography carrier is a temperature region in which the carrier has adsorptivity with a specific physiologically active substance, and is a physiologically active substance that can bind to a certain amount of carrier. This is a temperature region where the binding amount of the physiologically active substance is 50% or more with respect to the maximum binding amount.
  • the high temperature region in the first temperature-responsive chromatography carrier is a region where the carrier does not have an adsorptivity with a specific physiologically active substance, and is a physiologically active substance that can bind to a certain amount of the carrier. This is a region where the binding amount of the physiologically active substance is less than 50% with respect to the maximum binding amount.
  • a specific method for specifying the low temperature region and the high temperature region in the first temperature-responsive chromatography carrier is performed by the following procedure. 1.
  • the physiologically active substance is bound to the first temperature-responsive chromatographic carrier at less than 5 ° C., 10 ° C., respectively, and thereafter at temperatures set at intervals of 10 ° C. to just below the temperature at which the physiologically active substance is denatured.
  • the temperature is raised to just below the temperature at which the specific physiologically active substance is denatured, the bound physiologically active substance is eluted, and the physiologically active substance is quantified. 3.
  • the elution amount of the physiologically active substance is plotted against the temperature at which the protein is adsorbed, and 50% of the maximum value of the binding amount (elution amount) of the physiologically active substance and the intersection of the lines connecting the plots (hereinafter referred to as “50”).
  • the temperature region having a binding amount of 50% or more of the physiologically active substance at the boundary is referred to as a low temperature region in the first temperature-responsive chromatography carrier, and the binding amount of the physiologically active substance of less than 50%.
  • a temperature region having a temperature is defined as a high temperature region in the first temperature-responsive chromatography carrier.
  • the high temperature region in the second temperature-responsive chromatography carrier is a temperature region in which the carrier has adsorptivity with a specific physiologically active substance, and is a physiologically active substance that can bind to a certain amount of carrier. This is a temperature region where the binding amount of the physiologically active substance is 50% or more with respect to the maximum binding amount.
  • the low temperature region in the second temperature-responsive chromatography carrier is a region where the carrier does not have an adsorptivity to a specific physiologically active substance, and is a physiologically active substance that can bind to a certain amount of carrier. This is a region where the binding amount of the physiologically active substance is less than 50% with respect to the maximum binding amount.
  • a specific method for specifying the high temperature region and the low temperature region in the second temperature-responsive chromatography carrier is performed by the following procedure. 1.
  • the physiologically active substance is bound to the second temperature-responsive chromatographic carrier at each temperature set at intervals of 10 ° C. from just below the temperature at which the physiologically active substance is denatured to less than 5 ° C.
  • the temperature is lowered to less than 2.5 ° C., the bound physiologically active substance is eluted, and the physiologically active substance is quantified.
  • the elution amount of the physiologically active substance is plotted against the temperature at which the protein is adsorbed, and 50% of the maximum value of the binding amount (elution amount) of the physiologically active substance and the intersection of the lines connecting the plots (hereinafter referred to as “50”).
  • the temperature region having a binding amount of 50% or more of the physiologically active substance at the boundary is referred to as the high temperature region in the second temperature-responsive chromatography carrier, and the binding of the physiologically active substance of less than 50%.
  • the temperature region having the quantity is defined as the low temperature region in the second temperature-responsive chromatography carrier.
  • the low temperature region where the first temperature-responsive chromatography carrier adsorbs the physiologically active substance is, for example, 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., and most preferably 2 ° C. or higher and lower than 13 ° C.
  • the high temperature region where the first temperature-responsive chromatography carrier desorbs the physiologically active substance is, for example, 20 ° C. or higher and lower than 60 ° C., preferably 25 ° C. or higher and lower than 50 ° C., and most preferably 30 ° C. or higher and lower than 45 ° C. .
  • the high temperature region where the second temperature-responsive chromatography carrier adsorbs the physiologically active substance is, for example, 20 ° C. or more and less than 60 ° C., preferably 25 ° C. or more and less than 50 ° C., and most preferably 30 ° C. or more and less than 45 ° C.
  • the low temperature region where the second temperature-responsive chromatography carrier desorbs the physiologically active substance is, for example, 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., most preferably 2 ° C. or higher and lower than 13 ° C. .
  • the method for purifying a physiologically active substance includes at least A-1) a step of adsorbing the physiologically active substance to the first temperature-responsive chromatography carrier in a low temperature region, and A-2) adsorbing the physiologically active substance.
  • the temperature at which the physiologically active substance is adsorbed on the first temperature-responsive chromatography carrier is equal to or higher than the temperature at which the solution containing the physiologically active substance is frozen. It is preferably within ⁇ 30 ° C. from the 50% binding temperature of the chromatography carrier, more preferably within ⁇ 25 ° C., and most preferably within ⁇ 20 ° C. If the temperature when adsorbing the physiologically active substance to the first temperature-responsive chromatography carrier is low, the adsorptivity of the physiologically active substance can be increased, but the pressure in the column tends to increase. It is preferably within ⁇ 30 ° C. from the 50% binding temperature of the temperature-responsive chromatography carrier.
  • the temperature at which the physiologically active substance is desorbed from the first temperature-responsive chromatography carrier is within + 30 ° C. from the 50% binding temperature of the first temperature-responsive chromatography carrier. More preferably, it is within + 25 ° C, most preferably within + 20 ° C. If the temperature at which the physiologically active substance is desorbed from the first temperature-responsive chromatography carrier is high, the desorbability of the physiologically active substance is increased and the recovery rate can be increased, but the physiologically active substance is decomposed by heat. There is also a tendency to deactivate.
  • the temperature at which the physiologically active substance is adsorbed on the second temperature-responsive chromatography carrier is within + 30 ° C. from the 50% binding temperature of the second temperature-responsive chromatography carrier. Is more preferable, more preferably within + 25 ° C, and most preferably within + 20 ° C.
  • the higher the temperature at which the physiologically active substance is adsorbed to the second temperature-responsive chromatography carrier the higher the detachability of the physiologically active substance and the recovery rate, but the physiologically active substance is decomposed and lost by heat. There is also a tendency to live.
  • the temperature at which the physiologically active substance is adsorbed on the second temperature-responsive chromatography carrier is the same as that when the physiologically active substance is desorbed from the first temperature-responsive chromatography carrier.
  • the temperature is preferably below the temperature, and most preferably within the range of 0 to -5 ° C.
  • the temperature at which the physiologically active substance is desorbed from the second temperature-responsive chromatography carrier is equal to or higher than the temperature at which the solution containing the physiologically active substance is frozen. It is preferably within ⁇ 40 ° C., more preferably within ⁇ 35 ° C., and most preferably within ⁇ 30 ° C. from the 50% binding temperature of the hydrophilic chromatography carrier. If the temperature at which the physiologically active substance is adsorbed to the second temperature-responsive chromatography carrier is low, the detachability of the physiologically active substance can be increased and the recovery rate can be increased, but the pressure in the column tends to increase. Therefore, it is preferably within ⁇ 40 ° C. from the 50% binding temperature of the second temperature-responsive chromatography carrier.
  • the 50% binding temperature of the second temperature-responsive chromatography carrier is + 20 ° C. or less from the 50% binding temperature of the first temperature-responsive chromatography carrier. If the 50% binding temperature of the second temperature-responsive chromatography carrier is higher than the 50% binding temperature of the first temperature-responsive chromatography carrier by more than 20 ° C., the physiological response from the first temperature-responsive chromatography carrier Since the temperature at which the active substance is desorbed must be set high, the physiologically active substance tends to decompose and deactivate due to heat.
  • the pH condition of the solution containing the physiologically active substance is from 4 to 8 and the fluctuation range is within 1 until the steps A-1) to B-3).
  • the pH of a solution containing a physiologically active substance becomes acidic or alkaline, the physiologically active substance tends to be deactivated or aggregated.
  • the bioactive substance purification kit includes at least the first and second temperature-responsive chromatography carriers described above.
  • the temperature Th1 of the high temperature region where the first temperature-responsive chromatography carrier desorbs the physiologically active substance, and the temperature of the high temperature region where the second temperature-responsive chromatography carrier has the property of adsorbing the physiologically active substance Th2 Th1 -10 (° C) ⁇ Th2 ⁇ Th1 + 10 (° C) Have the relationship.
  • the temperature Th2 of the high temperature region in which the second temperature-responsive chromatography carrier adsorbs the physiologically active substance is the temperature Th2 of the high temperature region in which the first temperature-responsive chromatography carrier has the property of desorbing the physiologically active substance.
  • virus inactivation methods include a low pH method, a UV irradiation method, a surfactant treatment (SD treatment), a heat treatment, and a high pressure treatment.
  • the virus inactivation step can be inserted before step A-1), after step A-3), before step B-1) and after step B-3).
  • the low pH method is preferably used because it is easy to operate, but it is preferably carried out under conditions that do not cause inactivation or aggregation of the physiologically active substance.
  • the pH can be 3 to 5 and the treatment time can be 1 to 240 minutes.
  • the temperature-responsive ion exchange chromatography carrier a temperature-responsive ion exchanger in which a copolymer containing N-isopropylacrylamide or the like is fixed to the substrate surface can be used.
  • the copolymer has at least an ion exchange group.
  • the temperature-responsive ion exchanger according to this embodiment includes a monomer mixture composed of a monomer having an ion exchange group and / or an ion exchange group introduction precursor monomer and an N-isopropylacrylamide monomer by a surface graft polymerization method. It is formed by polymerization on the surface.
  • the shape of the substrate used in the temperature-responsive ion exchange chromatography carrier according to this embodiment is not particularly limited, and examples thereof include beads, flat plates, and tubes.
  • beads beads having various particle diameters are available and are not particularly limited, but the particle diameter is, for example, 1 to 300 ⁇ m, preferably 10 to 200 ⁇ m, more preferably 20 to 150 ⁇ m. is there. If the particle size is 1 ⁇ m or less, consolidation of beads tends to occur in the column, and thus processing at a high flow rate tends to be difficult. On the other hand, when the particle size is 300 ⁇ m or more, the gap between the beads becomes large, and the solution tends to leak when the biopolymer is adsorbed.
  • the base material used in this embodiment has a plurality of pores, for example.
  • the pore diameter is not particularly limited, but is, for example, 5 to 1000 nm, preferably 10 to 700 nm, and more preferably 20 to 500 nm.
  • the pore diameter is 5 nm or less, the molecular weight of the biopolymer that can be separated tends to be low.
  • the pore diameter is 1000 nm or more, the surface area of the substrate is reduced, and the antibody binding capacity tends to be reduced.
  • the material of the substrate is not particularly limited, but when it is in the form of beads, glass, silica, polystyrene resin, methacrylic resin, crosslinked agarose, crosslinked dextran, crosslinked polyvinyl alcohol, crosslinked cellulose, and the like can be used.
  • a temperature-responsive polymer having an ion exchange group is immobilized on the base material.
  • an atom transfer radical polymerization initiator is immobilized on the surface of the substrate, and a temperature responsive polymer is grown from the initiator in the presence of a catalyst. Is generated, and a “radiation graft polymerization method” in which a temperature-responsive polymer is allowed to grow using the generated radical as a starting point is not particularly limited.
  • an immobilization method there is an “atom transfer radical polymerization method” which is a surface living radical polymerization method.
  • the “atom transfer radical polymerization method” can be suitably used because the polymer can be fixed at a high density on the substrate surface.
  • the initiator used at that time is not particularly limited, but the substrate has a hydroxyl group as in this embodiment.
  • 2-mer chains are grown from this initiator.
  • the catalyst at that time is not particularly limited, and examples of the copper halide (CuIX) include CuICl and CuIBr.
  • the ligand complex for the copper halide is not particularly limited, but tris (2- (dimethylamino) ethyl) amine (Me6TREN), N, N, N ′′, N ′′ -pentamethyldiethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (HMTETA), 1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me4Cyclam), And bipyridine.
  • any means can be adopted to generate radicals on the substrate, but ionization is required to generate uniform radicals on the entire substrate. Irradiation with actinic radiation is preferred.
  • types of ionizing radiation ⁇ rays, electron beams, ⁇ rays, neutron rays and the like can be used. However, electron beams or ⁇ rays are preferable for implementation on an industrial scale.
  • the ionizing radiation is obtained from radioactive isotopes such as cobalt 60, strontium 90, and cesium 137, or by an X-ray imaging apparatus, an electron beam accelerator, an ultraviolet irradiation apparatus, or the like.
  • the irradiation dose of ionizing radiation is, for example, 1 kGy or more and 1000 kGy or less, preferably 2 kGy or more and 500 kGy or less, and more preferably 5 kGy or more and 200 kGy or less. If the irradiation dose is less than 1 kGy, radicals tend not to be generated uniformly. Further, when the irradiation dose exceeds 1000 kGy, the physical strength of the substrate tends to be lowered.
  • the graft polymerization method by irradiation with ionizing radiation generally involves generating a radical on the substrate and then bringing it into contact with the reactive compound, followed by a pre-irradiation method, and contacting the film with the reactive compound on the substrate. It is roughly divided into a simultaneous irradiation method for generating radicals. In this embodiment, any method can be applied, but a pre-irradiation method with less oligomer formation is preferred.
  • the solvent used at the time of polymerization is not particularly limited as long as it can uniformly dissolve the reactive compound.
  • solvents include alcohols such as ethanol, isopropanol, and t-butyl alcohol, ethers such as diethyl ether and tetrahydrofuran, ketones such as acetone and 2-butanone, water, and mixtures thereof.
  • the polymer coated on the substrate surface has a temperature-responsive monomer such as N-isopropylacrylamide.
  • Poly (N-isopropylacrylamide) is known to have a lower critical temperature at 32 degrees. Since the carrier introduced on the polymer surface greatly changes the hydrophilic / hydrophobic surface properties at the critical temperature, when used by grafting or coating on the surface of the base material (filler) of the chromatography carrier, The holding force for the sample can be obtained by the temperature. As a result, the retention behavior can be controlled by temperature without changing the composition of the eluate.
  • hydrophilic comonomers such as acrylamide, methacrylic acid, acrylic acid, dimethylacrylamide, and vinylpyrrolidone, which are more hydrophilic monomers than isopropylacrylamide, are combined with N-isopropylacrylamide. It can be prepared by polymerizing.
  • the lower critical temperature is desired to be 32 ° C. or lower, it can be prepared by copolymerization with a hydrophobic comonomer such as styrene, alkyl methacrylate, alkyl acrylate or the like which is a hydrophobic monomer.
  • Ion exchange groups can be broadly classified into strong anion exchange groups, weak anion exchange groups, strong cation exchange groups, and weak cation exchange groups.
  • the most appropriate ion exchange group is selected according to the type of physiologically active substance to be purified. There is a need to. Generally, when adsorbing basic proteins such as immunoglobulin, lysozyme, hemoglobin ⁇ chain, catalase, annexin, and ezrin, it is preferable to select a cation exchange group, and when adsorbing acidic proteins such as albumin It is preferable to select an anion exchange group. In the present embodiment, a case where a strong cation exchange group is selected as the ion exchange group and the antibody is adsorbed will be described in detail, but the present embodiment is not limited to this.
  • a polymer having a strong cation exchange group such as a sulfonic acid group will be described below as a polymer coated on the substrate surface.
  • a method for providing a strong cation exchange group is not particularly limited, but as a first method, when synthesizing a temperature-responsive polymer chain to be coated on the surface of a carrier, a method including copolymerization including a monomer having a strong cation exchange group is provided.
  • the monomer having a sulfonic acid group include (meth) acrylamide alkyl sulfonic acid, which is a structural unit of a polymer having sulfonic acid.
  • the “strong cation exchange group-introduced precursor” may include “a strong cation exchange group precursor”.
  • the “precursor of a strong cation exchange group” is, for example, a strong cation exchange group with a protective group. Examples of the monomer having a sulfonic acid group precursor include phenyl vinyl sulfonate, but the present embodiment is not limited thereto.
  • a strong cation exchange group can be given after copolymerization including a monomer having a functional group that can give a strong cation exchange group.
  • the method of converting a functional group into a sulfonic acid group is mentioned.
  • the monomer having a functional group capable of imparting a strong cation exchange group include glycidyl methacrylate.
  • a monomer composition in which the ratio of the monomer having a strong cation exchange group and / or the strong cation exchange group-introduced precursor monomer to N-isopropylacrylamide is 0.01 to 5 mol% is used as the surface graft weight. Polymerize by law. The ratio is preferably 0.1 to 4 mol%, more preferably 0.2 to 3 mol%, still more preferably 0.3 to 2 mol%, and most preferably 0.5 to 1.5 mol%. If the ratio exceeds 5 mol%, the amount of strong cation exchange groups relative to N-isopropylacrylamide in the copolymer will be excessive.
  • the amount of immunoglobulin adsorbed on the temperature-responsive adsorbent increases, it tends to be difficult to elute the adsorbed immunoglobulin due to temperature change.
  • the ratio is less than 0.01 mol%, the amount of strong cation exchange groups introduced is too small, and the amount of immunoglobulin adsorbed tends to decrease.
  • the polymer coated on the surface of the substrate causes hydration and dehydration by changing the temperature.
  • the temperature range is 0 ° C. or more and less than 80 ° C., preferably 5 ° C. or more and less than 50 ° C. More preferably, it is 10 ° C or higher and lower than 45 ° C. If the temperature exceeds 80 ° C., the mobile phase is water, and thus evaporation occurs and the workability tends to deteriorate. On the other hand, if it is lower than 0 ° C., the mobile phase tends to freeze.
  • the temperature-responsive ion exchange chromatography carrier obtained by this embodiment is attached to a normal liquid chromatography apparatus and used as a liquid chromatography system.
  • the method of loading the temperature-responsive ion exchange chromatography carrier with temperature is not particularly limited.
  • an aluminum block, water bath, air layer, jacket or the like having a predetermined temperature is attached to the temperature-responsive ion exchange chromatography carrier.
  • it may be attached.
  • the separation method using the temperature-responsive adsorbent of the present embodiment is not particularly limited, but as an example, the target biopolymer is once adsorbed on the obtained temperature-responsive liquid chromatography carrier, Thereafter, a method of utilizing the catch-and-release method, in which the adsorbed biopolymer is released by changing the temperature of the carrier surface by changing the temperature, can be mentioned.
  • the mass to be adsorbed may or may not exceed the amount that can be adsorbed on the carrier.
  • this is a purification method in which the adsorbed solute is released by once adsorbing and then changing the temperature and changing the characteristics of the support surface.
  • the temperature range in which the temperature-responsive ion exchange chromatography carrier, which is the second temperature-responsive chromatography carrier, adsorbs the physiologically active substance is, for example, 20 ° C. or more and less than 60 ° C., preferably 25 ° C. or more and 50 ° C. Less, most preferably in a high temperature region of 30 ° C. or higher and lower than 45 ° C.
  • the temperature range where the temperature-responsive ion exchange chromatography carrier desorbs the physiologically active substance is, for example, a low temperature range of 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., most preferably 2 ° C. or higher and lower than 13 ° C. is there.
  • the chromatography shown in the present embodiment may use a buffer solution as a mobile phase and does not require an organic solvent.
  • the buffer solution is an aqueous solution containing inorganic salts, and specifically includes a phosphate buffer solution, a Tris buffer solution, an acetate buffer solution, and the like. It is not limited.
  • the concentration of the inorganic salt is, for example, 1 to 50 mmol / L, preferably 3 to 40 mmol / L, and more preferably 5 to 30 mmol / L. If the concentration of the inorganic salt in the mobile phase is lower than 1 mmol / L, the activity of the physiologically active substance as a solute tends to be impaired.
  • the degree of dissociation of ion-exchange groups on the surface of the temperature-responsive adsorbent increases, and the solute is strongly adsorbed on the surface of the temperature-responsive adsorbent, making it difficult to remove the solute from the surface of the carrier in subsequent operations. There is a tendency.
  • the concentration of inorganic salts is higher than 50 mmol / L, the degree of dissociation of ion exchange groups on the surface of the temperature-responsive adsorbent becomes low, making it difficult to maintain the solute on the surface of the carrier, and finally separating the solute. Tend to be difficult.
  • the neutral buffer used in the present embodiment has, for example, a pH of 4.0 to 7.5, preferably 4.5 to 7.0, more preferably 5.0 to 6.5. is there.
  • the pH of the buffer solution is higher than 7.5, the immunoglobulin (isoelectric point 7.5 to 10) is negatively charged. Therefore, charge repulsion occurs with the strong cation exchange group of the temperature-responsive adsorbent of this embodiment. , The adsorption capacity tends to decrease extremely.
  • the pH is lower than 4.0, the immunoglobulin is denatured and tends to cause a decrease in activity and quality such as formation of aggregates.
  • the protein to be purified is not particularly limited, but a basic protein is preferable when a carrier surface having a strong cation exchange group is used.
  • basic proteins include immunoglobulins, lysozyme, hemoglobin ⁇ chain, catalase, annexin, and ezrin.
  • a carrier having a strong cation exchange group can be particularly suitably used for purification of immunoglobulin.
  • the shape of the support used in the temperature-responsive affinity chromatography carrier according to the present embodiment is not particularly limited, and may be, for example, a flat membrane shape, a hollow fiber shape, or a bead shape.
  • a hollow fiber-like support can be suitably used because it can be easily molded into a module and has a large membrane area that can be filled per module container.
  • a bead-shaped material generally has a larger surface area per volume than a film-shaped material and can adsorb a large amount of antibody, so that it can be suitably used.
  • the material of the support is not particularly limited, but when the carrier is in the form of a film, a polymer material capable of forming a porous film can be suitably used.
  • a polymer material capable of forming a porous film
  • olefin resins such as polyethylene and polypropylene
  • polyester resins such as polyethylene terephthalate and polyethylene terephthalate
  • polyamide resins such as nylon 6 and nylon 66
  • fluorine-containing resins such as polyvinylidene fluoride and polychlorotrifluoroethylene
  • polystyrene polysulfone
  • Noncrystalline resins such as polyethersulfone and polycarbonate
  • the support material may be glass, silica, polystyrene resin, methacrylic resin, crosslinked agarose, crosslinked dextran, crosslinked polyvinyl alcohol, crosslinked cellulose, and the like.
  • Crosslinked polyvinyl alcohol and crosslinked cellulose are highly hydrophilic and can be suitably used because they can suppress the adsorption of impurity components.
  • the support used in this embodiment has, for example, a plurality of pores.
  • the pore diameter is not particularly limited, but is, for example, 5 to 1000 nm, preferably 10 to 700 nm, and more preferably 20 to 500 nm. If the pore diameter is 5 nm or less, the molecular weight of the separable antibody tends to be low. Further, when the pore diameter is 1000 nm or more, the surface area of the substrate is reduced, and the antibody binding capacity tends to be reduced.
  • any coupling group may be introduced into the support.
  • the coupling group include a carboxyl group, carboxyl group, cyanogen bromide activation group, hydroxyl group, epoxy group, aldehyde group, and thiol group activated with N-hydroxysuccinimide (NHS).
  • NHS activity that can be coupled with a primary amino group Preferred are a carboxyl group, a carboxyl group, a cyanogen bromide active group, an epoxy group, and a formyl group.
  • a carboxyl group activated with NHS is preferably used because no other chemicals are required during the coupling reaction, and the reaction is rapid and forms a strong bond.
  • any method may be used for introducing the coupling group into the support, but a spacer is generally introduced between the support and the coupling group.
  • Methods for introducing coupling groups are disclosed in various literatures.
  • a graft polymer chain having a coupling group at the terminal and / or side chain may be introduced into the support.
  • a graft polymer chain having a coupling group By introducing a graft polymer chain having a coupling group into the support, it is possible to control the density of the coupling group as desired.
  • a polymer chain having a coupling group is grafted to the support, or a polymer chain having a precursor functional group that can be converted into a coupling group is grafted to the support, and then the grafted precursor functional group is cupped. It may be converted to a ring group.
  • the graft polymer chain can be introduced by any method.
  • a polymer chain may be prepared in advance and coupled to a support. Further, the graft chain may be polymerized directly on the support by the “living radical polymerization method” or the “radiation graft polymerization method”.
  • the “radiation grafting method” can be suitably used because there is no need to introduce a reaction initiator into the support in advance, and there are a variety of applicable supports.
  • any means can be adopted for generating radicals on the support, but in order to generate uniform radicals on the entire support, ionizing radiation can be used. Irradiation is preferred. As types of ionizing radiation, ⁇ rays, electron beams, ⁇ rays, neutron rays and the like can be used. However, electron beams or ⁇ rays are preferable for implementation on an industrial scale.
  • the ionizing radiation is obtained from radioactive isotopes such as cobalt 60, strontium 90, and cesium 137, or from an X-ray imaging apparatus, an electron beam accelerator, an ultraviolet irradiation apparatus, or the like.
  • the irradiation dose of ionizing radiation is, for example, preferably 1 kGy or more and 1000 kGy or less, more preferably 2 kGy or more and 500 kGy or less, and further preferably 5 kGy or more and 200 kGy or less. If the irradiation dose is less than 1 kGy, radicals tend not to be generated uniformly. Further, when the irradiation dose exceeds 1000 kGy, the physical strength of the support tends to be lowered.
  • a pre-irradiation method in which the radical is then contacted with a reactive compound, and the support in a state where the support is in contact with the reactive compound.
  • a simultaneous irradiation method for generating radicals in this embodiment, any method can be applied, but a pre-irradiation method with less oligomer formation is preferred.
  • the solvent used in the graft polymerization is not particularly limited as long as the reactive compound can be uniformly dissolved.
  • a solvent include alcohols such as ethanol, isopropanol and t-butyl alcohol; ethers such as diethyl ether and tetrahydrofuran; ketones such as acetone and 2-butanone; water, or a mixture thereof.
  • examples of the monomer having a coupling group used for graft polymerization include monomers such as acrylic acid and methacrylic acid when a carboxyl group is used as the coupling group.
  • a primary amino group is used as a coupling group, allylamine and the like can be mentioned.
  • an epoxy group is mentioned.
  • a monomer having a precursor functional group that can be converted into a coupling group may be grafted to a support, and then the grafted precursor functional group may be converted into a coupling group.
  • Glycidyl methacrylate (GMA) having an epoxy group can be converted into various functional groups using ring-opening reactions of various epoxy groups, and thus can be suitably used industrially.
  • the epoxy group of GMA is hydrolyzed into a diol, and a cyclic acid anhydride is subjected to a ring-opening half esterification reaction with a hydroxyl group derived from the diol. It is possible to form a carboxyl group derived from a cyclic acid anhydride (ring-opening half esterification reaction).
  • the cyclic acid anhydride is preferably succinic anhydride or glutaric anhydride, but is not limited thereto.
  • the catalyst used in the ring-opening half esterification reaction is not particularly limited as long as it promotes this reaction, and specific examples include triethylamine, isobutylethylamine, pyridine, 4-dimethylaminopyridine, and the like. 4-Dimethylaminopyridine is preferred, and 4-dimethylaminopyridine is most preferred in view of reaction rate and yield.
  • the ring-opening half esterification reaction is preferably performed in an inert organic solvent such as toluene to which the above catalyst is added.
  • the NHS activation reaction is a step of converting a carboxyl group formed by the ring-opening half esterification reaction into an active ester. Since the active ester is more reactive than the carboxyl group, the active esterification step is preferably performed when it is desired to quickly immobilize the temperature-responsive protein A on the carrier.
  • the active ester serves to bind the hydrophilic compound and the substance to be immobilized by a covalent bond.
  • the active ester means a chemical structure of R—C ( ⁇ O) —X.
  • X is a leaving group such as, but not limited to, halogen, N-hydroxysuccinimide group or derivative thereof, 1-hydroxybenzotriazole group or derivative thereof, pentafluorophenyl group, and paranitrophenyl group.
  • N-hydroxysuccinimide ester is desirable in terms of reactivity, safety and production cost. Conversion of the carboxyl group to an N-hydroxysuccinimide ester is achieved by reacting the carboxyl group with N-hydroxysuccinimide and carbodiimide simultaneously.
  • carbodiimide means an organic compound having a chemical structure of —N ⁇ C ⁇ N—, such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
  • concentration of N-hydroxysuccinimide and carbodiimide is 1 to 100 mmol / L
  • the reaction temperature is 0 ° C. or more and less than 100 ° C.
  • the reaction time is 2 minutes to 16 hours.
  • N, N'-dimethylformamide (DMF), toluene, or the like can be used as the reaction solvent.
  • the temperature-responsive affinity chromatography carrier comprises temperature-responsive protein A, which is protein A mutated so that the binding property to the antibody changes depending on temperature
  • the temperature-responsive protein A is: It can be prepared with reference to a patent document (WO2008 / 143199 pamphlet).
  • the coupling reaction between the NHS activated carboxyl group and the temperature-responsive protein A is performed as follows, for example. First, citrate buffer (pH 3.0 to 6.2), acetate buffer (pH 3.6 to 5.6), phosphate buffered saline (PBS, pH 5.8 to 8.5), or carbonate buffer A 0.1-100 mg / mL temperature-responsive protein A solution is prepared using a buffer solution that does not contain an amino group component such as a solution (pH 9.2 to 10.6). When this aqueous solution is brought into contact with the active ester surface, a functional group such as an amino group contained in the temperature-responsive protein A reacts with the active ester to form an amide bond.
  • citrate buffer pH 3.0 to 6.2
  • acetate buffer pH 3.6 to 5.6
  • phosphate buffered saline PBS, pH 5.8 to 8.5
  • carbonate buffer A 0.1-100 mg / mL temperature-responsive protein A solution is prepared using a buffer solution that does not contain an amino
  • temperature-responsive protein A is immobilized on the surface by covalent bonds.
  • the contact time may be set in the range of 2 minutes to 16 hours.
  • the washing solution is preferably a buffer solution containing about 0.5 mol / L of salt (NaCl) and about 0.1% of nonionic surfactant. This is because the temperature-responsive protein A that is physically adsorbed without being covalently bonded can be removed.
  • the unreacted carboxyl group or active ester is converted into a low molecular compound having an amino group and It is preferable to convert the carboxyl group or the active ester into a functional group having a lower reactivity by bonding. As a result, it is possible to prevent molecules such as impurities that are not subject to purification from being unintentionally immobilized on the surface of the carrier.
  • the functional group at the end of the temperature-responsive protein A fixing carrier is an active ester, this operation is preferably performed.
  • the operation of reacting a low molecular weight compound having an amino group with an active ester group may be particularly described as “blocking”.
  • the surface of the carrier after reacting the carboxyl group or the active ester with the low molecular weight compound is hydrophilic. This is because a hydrophilic surface generally has an effect of suppressing nonspecific adsorption of a biological substance.
  • Non-limiting examples of such low molecular weight compounds include ethanolamine, trishydroxymethylaminomethane, and diglycolamine (IUPAC name: 2- (2-aminoethoxy) ethanol).
  • reaction temperature may be set in the range of 4 to 37 ° C. and the reaction time in the range of 2 minutes to 16 hours.
  • the temperature-responsive protein A-immobilized carrier is stored at a low temperature of about 2-10 ° C. with a neutral solution in the pH range of 4-8 as a storage solution.
  • a neutral solution in the pH range of 4-8 as a storage solution.
  • 20% ethanol is preferable in consideration of antibacterial properties.
  • Temperature-responsive protein A has a characteristic that it can bind an antibody at a low temperature and can elute the antibody at a temperature higher than the temperature at the time of binding. It is preferable that the temperature at which the characteristics of the temperature-responsive protein A change in advance is confirmed, and the antibody is adsorbed and desorbed by changing the temperature so as to sandwich the temperature.
  • the temperature range in which the antibody is adsorbed to the temperature-responsive protein A is, for example, a low temperature range of 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., most preferably 2 ° C. or higher and lower than 13 ° C.
  • the temperature at which the antibody is desorbed from the temperature-responsive protein A is, for example, a high temperature region of 20 ° C. or higher and lower than 60 ° C., preferably 25 ° C. or higher and lower than 50 ° C., most preferably 30 ° C. or higher and lower than 45 ° C.
  • the antibody-containing mixture is transformed with a hybridoma that produces the antibody, a myeloma cell such as NSO, and a gene encoding the antibody.
  • Culture cells such as animal cells and yeast that can be expressed and produced are included.
  • these culture supernatants are clarified when purification by the method of this embodiment is performed. The clarification may be performed, for example, by filtration with a 0.2 ⁇ m membrane filter.
  • the antibody to be purified in this embodiment is not limited, and examples thereof include human antibodies, non-human animal antibodies such as mice, cows, goats, sheep and other ungulates, chimeric antibodies between humans and non-human animals, and non-human animals.
  • examples include humanized antibodies obtained by humanizing antibodies, and preferably human antibodies. More preferred is a human monoclonal antibody.
  • the class and subclass of the antibody are not limited, and antibodies of any class and subclass can be purified in the present embodiment, but IgG is preferable, and IgG1, IgG2, and IgG4 among them are preferable.
  • the antibody in the amino acid sequence of the heavy chain constant region of the antibody, at least one amino acid of the naturally occurring heavy chain constant region is deleted, or at least one of the naturally occurring heavy chain constant region amino acids. It may be an antibody in which one amino acid is substituted with another amino acid or at least one amino acid is added to a naturally occurring heavy chain constant region. Furthermore, the antibody may be covalently or coordinated with another compound.
  • the temperature-responsive chromatography carrier When the temperature-responsive chromatography carrier is fixed to a bead-like carrier, it may be packed into a commercially available empty column or an empty column prepared from a glass tube.
  • a commercially available empty column with a jacket (trade name: XK column, GE Healthcare Japan) can be suitably used because the temperature of the column itself can be arbitrarily controlled by controlling the temperature of the jacket circulating water. I can do it.
  • the temperature-responsive chromatography carrier When the temperature-responsive chromatography carrier is in the form of a membrane, it may be fixed to a commercially available membrane holder or processed into an arbitrary module shape according to each shape.
  • the column 20 provided with a temperature-responsive cation exchange chromatography carrier as a chromatography carrier is connected in series via a pipe 104.
  • the length of the pipe 104 between the columns is preferably as short as possible and further kept warm in order to minimize the temperature change due to heat radiation.
  • a three-way valve for switching the flow path may be installed in the pipe 104 between the columns, and the drain valve 60 may be disposed.
  • the drain valve 60 is a waste liquid containing contaminants flowing out from the temperature-responsive affinity chromatography carrier column 10 when supplying the antibody-containing mixture to the temperature-responsive affinity chromatography carrier column 10 including the temperature-responsive protein A. Is used to prevent the waste liquid containing impurities from being supplied to the temperature-responsive cation exchange chromatography support column 20.
  • the mixture solution containing the antibody is cooled to a temperature that is adsorbed to temperature-responsive protein A, and then temperature-responsive protein A.
  • a temperature-responsive affinity chromatography carrier column 10. it is necessary to confirm in advance the temperature at which the temperature-responsive protein A is binding to the antibody, and to adjust the temperature of the mixture solution containing the antibody to that temperature.
  • the temperature at which the antibody is adsorbed to the temperature-responsive protein A is 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., most preferably 2 ° C. or higher and lower than 13 ° C.
  • the mixture solution containing the antibody may contain impurities such as protease and may be stored at a low temperature.
  • the storage temperature of the mixture containing the antibody is within the temperature range where the temperature-responsive protein A has a binding property to the antibody, the antibody may be directly adsorbed to the temperature-responsive protein A.
  • a heat exchanger is disposed immediately upstream of the temperature-responsive protein A column, and the temperature of the mixture solution containing the antibody is continuously changed while loading the antibody-containing mixture solution onto the temperature-responsive protein A column. It is also possible to adjust. It is also possible to adjust the temperature of the mixture solution containing the antibody by immersing the temperature-responsive protein A column in a constant temperature water bath adjusted to a predetermined temperature.
  • the antibody-containing mixture is further immersed by immersing the temperature-responsive protein A column in a constant temperature water bath adjusted to a predetermined temperature. It is also possible to adjust the temperature of the solution.
  • the antibody-containing mixture solution is supplied to the temperature-responsive affinity chromatography carrier column 10 to bind the antibody to the temperature-responsive protein A, and then the phosphate buffer and the Tris-HCl buffer.
  • the carrier column 10 may be washed with a buffer solution such as By washing with the buffer solution, the impurities remaining on the carrier column 10 can be washed to improve the purity of the recovered antibody.
  • the buffer used for washing is used after the temperature-responsive protein A has been adjusted to a temperature range in which it binds to the antibody.
  • the antibody bound to temperature-responsive protein A can be eluted with a high-temperature buffer.
  • the temperature of the buffer solution when eluting the antibody is in a temperature range where the temperature-responsive protein A loses its binding property to the antibody, and the temperature-responsive cation exchange chromatography carrier has a temperature range in which the antibody is binding.
  • the temperature of the buffer solution when eluting the antibody varies depending on the temperature-responsive cation exchange chromatography carrier to be used, but it is 20 ° C. or more and less than 60 ° C., preferably 25 ° C. or more and less than 50 ° C., most preferably 30 ° C. or more and less than 45 ° C. It is.
  • the antibody eluted from temperature-responsive protein A is supplied to the temperature-responsive cation exchange chromatography carrier column 20 while maintaining the temperature as it is. Bound to a temperature-responsive cation exchange chromatography support.
  • the antibody solution eluted from the temperature-responsive protein A can be temporarily stored in a tank or the like, but not to the extent that the temperature-responsive cation exchange chromatography carrier does not deviate from the temperature range in which the antibody-binding ability is bound. It is preferable to keep warm.
  • the antibody eluted from temperature-responsive protein A is subjected to chromatography steps such as gel filtration, anion exchange, chromatofocusing, affinity, group-specific affinity, hydrophobic interaction, reverse phase, etc.
  • the responsive cation exchange chromatography support column 20 may be supplied. If a temperature-responsive protein A column and a temperature-responsive cation exchange chromatography carrier column are not directly connected to the temperature-sensitive protein A column without a tank or other purification step, The eluted antibody is preferable because it is easy to keep the temperature so that the temperature-responsive cation exchange chromatography carrier does not deviate from the temperature range in which the temperature-responsive cation exchange chromatography carrier binds to the antibody.
  • the antibody bound to the temperature-responsive cation exchange chromatography carrier can be eluted with a low-temperature buffer.
  • the temperature of the buffer solution during elution is a temperature region in which the temperature-responsive cation exchange chromatography carrier loses its binding property to the antibody.
  • the temperature of the buffer varies depending on the temperature-responsive cation exchange chromatography carrier used, but is 0 ° C. or higher and lower than 20 ° C., preferably 1 ° C. or higher and lower than 15 ° C., and most preferably 2 ° C. or higher and lower than 13 ° C.
  • a heat exchanger immediately upstream of the temperature-responsive cation exchange column and continuously pass a buffer solution at a predetermined temperature. It is also possible to elute the antibody by immersing the temperature-responsive cation exchange column in a constant temperature water bath adjusted to a predetermined temperature. In addition to using a heat exchanger arranged immediately upstream of the temperature-responsive cation exchange column, the antibody is eluted by immersing the temperature-responsive cation exchange column in a constant temperature water bath adjusted to a predetermined temperature. Is also possible.
  • NHS activation reaction liquid (NHS 0.07 g, dehydrated isopropyl alcohol 45 mL, diisopropylcarbodiimide 0.09 mL) was permeated at a flow rate of 0.4 mL / min for 30 minutes.
  • the carboxyl group was NHS activated.
  • the column was washed by passing dehydrated isopropyl alcohol through the column at a flow rate of 0.4 mL / min for 30 minutes. The washed column was stored at 4 ° C. with dehydrated isopropyl alcohol enclosed.
  • temperature-responsive protein A was prepared with reference to the examples in the patent document (WO2008 / 143199 pamphlet). Further, 2 mL of ice-cooled 1 mmol / L hydrochloric acid was passed through a column in which the carboxyl group had been NHS activated to replace dehydrated isopropyl alcohol as a stock solution. Next, 30 mg of temperature-responsive protein A was dissolved in 1 mL of coupling buffer (0.2 mol / L phosphate buffer, 0.5 mol / L NaCl, pH 8.3), and the module was flown at a flow rate of 0.4 mL / min. For 4 hours. After a predetermined time, the coupling buffer was permeated through the column to wash and collect the temperature-responsive protein A that did not undergo a coupling reaction with the NHS active group.
  • coupling buffer 0.2 mol / L phosphate buffer, 0.5 mol / L NaCl, pH 8.3
  • Blocking 10 mL of blocking reaction solution (0.5 mol / L ethanolamine, 0.5 mol / L NaCl, pH 8.0) permeates through a column coupled with temperature-responsive protein A, and left at room temperature for 30 minutes. Residual NHS was blocked with ethanolamine. After the reaction, the column was washed with pure water and then stored at 4 ° C. in a state sealed in the column with 20% ethanol.
  • the 50% bonding temperature was calculated according to the procedure described above. As a result, a certain amount of temperature-responsive protein A can bind to 14.5 mg / ml of antibody when the environmental temperature (antibody binding temperature) is 2 ° C., and at 2 ° C., the maximum binding amount is increased. Indicated.
  • the 50% adsorption temperature was 15.0 ° C. In this case, the temperature of 15.0 ° C. or lower is the low temperature region described above, and the temperature higher than 15.0 ° C. is the high temperature region.
  • thermo-responsive cation exchange chromatography carrier Preparation of temperature-responsive cation exchange chromatography carrier
  • a bead-like temperature-responsive adsorbent having a sulfonic acid group was prepared by an atom transfer radical polymerization method. Details are as follows.
  • N-isopropylacrylamide (IPAAm, manufactured by Wako Pure Chemical Industries, Ltd.) 18.40 g, GMA 0.231 g, butyl methacrylate (BMA, manufactured by Tokyo Chemical Industry Co., Ltd.) 1.217 g, copper chloride I ( 0.085 g of CuCl, manufactured by Wako Pure Chemical Industries, Ltd.) and 0.012 g of copper chloride II (CuCl2, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in 42.8 mL of a 90% by volume isopropanol (IPA) aqueous solution. Nitrogen bubbling was performed for 30 minutes.
  • IPA isopropanol
  • the 50% bonding temperature was calculated according to the procedure described above. As a result, a certain amount of temperature-responsive cation exchange chromatography carrier can bind to 30.1 mg / ml antibody when the environmental temperature (antibody binding temperature) is 40 ° C. The amount of binding is shown.
  • the 50% adsorption temperature was 31.0 ° C. In this case, the temperature lower than 31.0 ° C. is the low temperature region described above, and the temperature higher than 31.0 ° C. is the high temperature region.
  • Antibody purification test 1) Adsorption and elution of antibody (blood donation Venoglobuly-IH, manufactured by Benesis Co., Ltd.) due to temperature change using a step chromatography system with temperature-responsive protein A (AKTA FPLC, manufactured by GE Healthcare Japan Co., Ltd.) A test was conducted. First, as shown in FIG. 1, a column 10 packed with a temperature-responsive protein A carrier and a temperature-responsive cation exchange chromatography carrier column 20 are composed of a container 30 containing a buffer solution, a container 40 containing an antibody solution, a pump 50, And pipes 101, 102, 103, 104.
  • AKTA FPLC temperature-responsive protein A
  • the temperature change operation of the column 10 is performed by temporarily stopping the pump 50 of the chromatography system, and the pipe 103 upstream of the column 10 (manufactured by GE Healthcare Japan, PEEK tubing, od. 1/16, id. 0). (5 mm, orange) 1 m was wound in a loop shape, the pipe 103 was immersed in a constant temperature water bath at a predetermined temperature together with the column 10, and then incubated for 10 minutes or more, and then the pump 50 of the chromatography system was started again. .
  • the adsorption and elution of the antibody by the column 10 packed with the temperature-responsive protein A carrier were performed under the following conditions.
  • Adsorption step ⁇ Antibody concentration: 2.5 mg / mL Adsorption buffer: 15 mmol / L phosphate buffer (pH 6.0) ⁇ Equilibration: 10 bead volume (using adsorption buffer) ⁇ Antibody load: 20 mL ⁇ Flow rate: 0.4mL / min ⁇ Bead volume: 0.59 mL ⁇ Adsorption temperature: 2 °C 1-2) Washing step ⁇ Washing buffer: Same as adsorption buffer ⁇ Flow rate: 0.4 mL / min ⁇ Washing temperature: 2 °C 1-3) Temperature elution step ⁇ Temperature elution buffer: Same as adsorption buffer ⁇ Flow rate: 0.4 mL / min ⁇ Permeate volume: 20 mL ⁇ Elution temperature: 40 °C
  • Adsorption step • Antibody solution load: 20 mL ⁇ Flow rate: 0.4mL / min -Column volume: 0.54 mL ⁇ Adsorption temperature: 40 °C 2-2) Washing step • Washing buffer: 15 mmol / L phosphate buffer (pH 6.0) ⁇ Flow rate: 0.4mL / min ⁇ Washing temperature: 40 °C 2-3) Temperature elution step ⁇ Temperature elution buffer: 15 mmol / L phosphate buffer (pH 6.0) ⁇ Flow rate: 0.4mL / min ⁇ Flow rate: 20mL ⁇ Elution temperature: 2 °C
  • the UV absorption (280 nm) of the fraction obtained from the temperature elution step of the temperature-responsive cation exchange chromatography support column 20 was measured, the antibody concentration was calculated from the following formula, and the temperature elution amount of immunoglobulin was calculated.
  • Antibody concentration (mg / mL) Absorbance at 280 nm / 14 ⁇ 10
  • Temperature elution amount (mg / mL) Immunoglobulin concentration of the temperature elution fraction x liquid volume of the temperature elution fraction / column volume
  • the aggregate ratio (monomer and fraction) of the fraction obtained from the temperature elution step of the temperature-responsive cation exchange chromatography carrier column 20 The weight ratio of the multimer).
  • the weight ratio between the monomer of the antibody and the multimer was measured by high performance liquid chromatography (LC-20A system manufactured by Shimadzu Corporation, column G3000SWXL manufactured by Tosoh Corporation), and the absorption peak area at a wavelength of 280 nm. It was calculated from the ratio.
  • Aggregate ratio (%) (Area ratio of multimers / Area ratio of monomers) ⁇ 100
  • the antibody contained in the temperature elution step of the temperature-responsive cation exchange chromatography carrier column 20 was 8 mg, and the aggregate ratio was 5.8% (the antibody before purification by the column 10 packed with the temperature-responsive protein A carrier). The aggregate ratio was 5.6%). From these results, it was possible to purify the antibody without increasing the aggregate ratio by using the temperature-responsive protein A carrier column 10 and the temperature-responsive cation exchange chromatography carrier column 20.
  • the antibody purified in Example 1 (temperature elution fraction of the temperature-responsive cation exchange chromatography support column 20) is directly permeated through the anion exchange chromatography support membrane 200, thereby anion exchange. Impurities were bound to the chromatography support to remove impurities from the solution.
  • the anion exchange chromatography carrier membrane 200 a hollow fiber membrane prepared according to Reference Example 1 of JP2010-241761 was used. The flow-through step by the anion exchange chromatography support membrane 200 was performed under the following conditions. Equilibration buffer: 15 mmol / L phosphate buffer (pH 6.0) ⁇ Equilibration: 10 beads volume ⁇ Flow rate: 0.4 mL / min Effective membrane volume: 0.53 mL ⁇ Adsorption temperature: Room temperature
  • the amount of antibody contained in the flow-through fraction of the anion exchange chromatography carrier membrane 200 was 8 mg, and the aggregate ratio was 5.8%. From this result, in addition to the temperature-responsive protein A carrier column 10 and the temperature-responsive cation exchange chromatography carrier column 20, the three-step purification using the anion exchange chromatography carrier membrane 200 does not increase the ratio of aggregates. The antibody could be purified.
  • the elution fraction was collected in a polypropylene centrifuge tube (capacity 50 mL) and placed in a refrigerator (4 ° C.). I saved it all day and night. Thereafter, as shown in FIG. 4, the temperature of the elution fraction was raised to the adsorption temperature of the temperature-responsive cation exchange chromatography carrier 20 and then the elution fraction was bound to the temperature-responsive cation exchange chromatography carrier 20. Purified the antibody according to Example 1.
  • the pipe 103 upstream of the column 20 (GE Healthcare Japan, PEEK tubing, od 1/16, id 0.5 mm, orange) 1 m is wound in a loop. It was carried out by immersing in a constant temperature water bath at 70 ° C.
  • the antibody contained in the temperature elution step of the temperature-responsive cation exchange chromatography carrier column 20 shown in FIG. 4 is 7 mg, and the aggregate ratio is 7.2% (before purification by the column 10 packed with the temperature-responsive protein A carrier).
  • the antibody aggregate ratio was 5.6%).
  • the antibody is thermally denatured in the process of raising the temperature with a heating medium of 70 ° C. in the looped pipe 103 shown in FIG. The aggregate ratio was increased.
  • the eluate obtained from the temperature-responsive protein A column 10 is supplied to the temperature-responsive cation exchange chromatography carrier column 20 while maintaining the temperature as it is. It was shown that the antibody can be purified efficiently.
  • the antibody purification method according to the present embodiment makes it possible to purify a useful physiologically active substance such as an antibody on an industrial scale while suppressing denaturation using a temperature-responsive chromatography carrier.

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  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de purification d'une substance physiologiquement active, le procédé faisant appel à un premier support de chromatographie pouvant adsorber la substance physiologiquement active dans une région de basse température et désorber la substance physiologiquement active dans une région de température élevée et à un second support de chromatographie pouvant adsorber la substance physiologiquement active dans la région de température élevée et désorber la substance physiologiquement active dans la région de basse température. Le procédé comprend : l'absorption de la substance physiologiquement active sur le premier support de chromatographie à une température se trouvant dans la région de basse température ; l'augmentation de la température du premier support de chromatographie sur lequel la substance physiologiquement active est adsorbée pour ainsi désorber la substance physiologiquement active du premier support de chromatographie à une température se trouvant dans la région de température élevée ; l'adsorption de la substance physiologiquement active sur le second support de chromatographie à une température se trouvant dans la région de température élevée ; et la diminution de la température du second support de chromatographie sur lequel la substance physiologiquement active est adsorbée pour ainsi désorber la substance physiologiquement active du second support de chromatographie à une température se trouvant dans la région de basse température.
PCT/JP2012/056319 2011-03-10 2012-03-12 Procédé de purification d'une substance physiologiquement active au moyen d'un support de chromatographie sensible à la température WO2012121409A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2014065276A1 (fr) * 2012-10-23 2014-05-01 旭化成メディカル株式会社 Procédé et dispositif pour le raffinage d'une substance par chromatographie en phase liquide
WO2014171437A1 (fr) * 2013-04-16 2014-10-23 旭化成メディカル株式会社 Procédé de purification d'une protéine d'anticorps
JP2016183967A (ja) * 2015-03-25 2016-10-20 株式会社日立ハイテクサイエンス ２次元液体クロマトグラフ分析装置および分析法
WO2016199550A1 (fr) * 2015-06-12 2016-12-15 株式会社日立ハイテクノロジーズ Matériau adsorbant, et appareil de purification d'anticorps mettant en œuvre ledit matériau adsorbant
CN109320603A (zh) * 2018-12-17 2019-02-12 杭州奕安济世生物药业有限公司 一种连续化提纯抗体的系统及方法

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WO2008143199A1 (fr) * 2007-05-21 2008-11-27 Nomadic Bioscience Co., Ltd. Nouveau polypeptide, matériel pour chromatographie d'affinité, et procédé de séparation et/ou de purification d'une immunoglobuline
JP2011041475A (ja) * 2009-08-19 2011-03-03 Asahi Kasei Medical Co Ltd 抗体製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008143199A1 (fr) * 2007-05-21 2008-11-27 Nomadic Bioscience Co., Ltd. Nouveau polypeptide, matériel pour chromatographie d'affinité, et procédé de séparation et/ou de purification d'une immunoglobuline
JP2011041475A (ja) * 2009-08-19 2011-03-03 Asahi Kasei Medical Co Ltd 抗体製造方法

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014065276A1 (fr) * 2012-10-23 2014-05-01 旭化成メディカル株式会社 Procédé et dispositif pour le raffinage d'une substance par chromatographie en phase liquide
WO2014171437A1 (fr) * 2013-04-16 2014-10-23 旭化成メディカル株式会社 Procédé de purification d'une protéine d'anticorps
JPWO2014171437A1 (ja) * 2013-04-16 2017-02-23 旭化成メディカル株式会社 抗体タンパク質の精製方法
US10400007B2 (en) 2013-04-16 2019-09-03 Asahi Kasei Medical Co., Ltd. Method for purifying antibody protein
JP2016183967A (ja) * 2015-03-25 2016-10-20 株式会社日立ハイテクサイエンス ２次元液体クロマトグラフ分析装置および分析法
WO2016199550A1 (fr) * 2015-06-12 2016-12-15 株式会社日立ハイテクノロジーズ Matériau adsorbant, et appareil de purification d'anticorps mettant en œuvre ledit matériau adsorbant
CN109320603A (zh) * 2018-12-17 2019-02-12 杭州奕安济世生物药业有限公司 一种连续化提纯抗体的系统及方法

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