JP5594113B2 - Method for producing crosslinked polymer particle, and crosslinked polymer particle - Google Patents

Description

  The present invention relates to a method for producing crosslinked polymer particles and crosslinked polymer particles.

In recent years, in the field of biopharmaceuticals represented by antibody drugs and the like, expression techniques for target substances such as proteins have been remarkably advanced. Accordingly, there is a demand for improvement in productivity in a purification process such as chromatography.
Gels made of polysaccharides such as agarose and cellulose, which are natural polymers, are widely used as carriers for chromatographic fillers. For example, agarose can be dissolved in water at a high temperature, and then forms a porous gel when cooled to a certain temperature (gelation point). Therefore, the aqueous phase in which agarose is dissolved is dispersed in an organic solvent to form droplets. After forming, agarose gel particles can be obtained by cooling to below the gel point.

  Regarding cellulose, as a method for dissolving and regenerating cellulose into porous gel particles, a method of dissolving in a calcium thiocyanate aqueous solution (Patent Document 1), to a lithium chloride solution of dimethylacetamide or N-methylpyrrolidone. There have been reported a method of undergoing dissolution (Patent Document 2), a method of undergoing dissolution in an ionic liquid (Non-Patent Document 1), and the like.

  Since such polysaccharide gel particles contain a large number of hydroxyl groups in the molecule, the surface thereof has high hydrophilicity. Therefore, basically, non-specific interaction with a biological substance does not occur, so that the target molecule tends to be obtained with a high degree of purification. In general, compared to a carrier made of silica or synthetic polymer, it has excellent binding capacity to biomolecules and resistance to alkaline solutions generally used for washing chromatography fillers.

  However, when polysaccharide gel particles are used as a carrier for chromatographic packing materials, the mechanical strength is generally inferior to that of synthetic polymers, and as a result, the particles deform in the column when used at high flow rates. Therefore, there is a problem that the flow path is lost, and as a result, the back pressure of the column tends to be increased and the consolidation tends to occur, resulting in poor flow resistance.

  The most common method for improving the mechanical strength and flow resistance of polysaccharide gel particles is to crosslink the polysaccharide gel particles after particle formation. Such crosslinking occurs between the hydroxyl groups inside the polysaccharide gel particles and is generally performed by using a crosslinking agent such as epichlorohydrin. Various improvements have been reported for the formation of crosslinks so far. For example, after crosslinking with a bifunctional or polyfunctional crosslinking agent having a chain length of 6-12, crosslinking is performed with a bifunctional crosslinking agent having a length of 2-5 chains. Thus, the mechanical strength of the crosslinked agarose gel particles can be improved (Patent Document 3), and the cellulose gel particles are crosslinked with a crosslinking agent having a hydroxyl group between functional groups, thereby providing an appropriate compressive stress. (Patent Document 4), and by sequentially adding a crosslinking agent and an alkaline solution to a suspension containing cellulose gel particles, the entire amount of the crosslinking agent is charged at once. It has been reported that cellulose gel particles having excellent flow resistance can be obtained (Patent Document 5).

  In addition, as a method of crosslinking the inside of the particles more efficiently, there is a method in which a functional group is previously introduced into a polysaccharide as a raw material to form particles, and then the functional groups are activated to crosslink between the polysaccharide chains. is there. According to this method, it is possible to perform crosslinking even in a fine structure in which it is difficult for a crosslinking agent to enter as compared with the method of crosslinking after particle formation, and therefore, higher mechanical strength is expected. For example, Patent Document 6 discloses a crosslinked agarose gel by reacting agarose with an epoxy group of allyl glycidyl ether to form gel particles, and then activating the allyl group of allyl glycidyl ether to crosslink with the polysaccharide. It is described that the mechanical strength of the particles can be improved. However, this technique requires a multi-step reaction process from modification of agarose to crosslinking, and the operation is complicated. In addition, since agarose and the epoxy group of allyl glycidyl ether are reacted in water, the epoxy group is inactivated by water in the reaction system, resulting in a low reaction efficiency.

  As described above, there are many technologies that can be used for the production of a packing material for chromatography using polymer particles as a carrier. However, as the biopharmaceutical market expands, the need for high-speed processing in the purification process of target substances is rapidly increasing. There is a growing need in the art for a method of producing a chromatographic packing material that has both higher flow resistance and binding capacity.

Japanese Patent No. 3601229 Japanese Patent No. 3663666 JP-A-60-39558 JP 2008-279366 A JP 2009-242770 A Japanese Patent No. 4081143

J. Chromatogr. A 1217 (2010), 1298-1304

  The problem to be solved by the present invention is that when it is used as a packing material for chromatography, it can be used at a high flow rate, that is, it has excellent flow rate resistance suitable for mass processing, and an appropriate ligand is introduced. In such a case, it is to provide a crosslinked polymer particle having a high binding capacity for a target molecule such as a protein and a production method thereof.

  As a result of intensive studies, the present inventors have made it possible to react a polymer dissolved in an ionic liquid with a cross-linking agent while dispersing the liquid droplets in an organic solvent having low compatibility with the ionic liquid. It has been found that when a suitable ligand is introduced, crosslinked polymer particles having a high binding capacity for proteins can be obtained and the above-mentioned problems can be solved.

That is, the present invention relates to the following 1) to 11).
1) A method for producing crosslinked polymer particles, comprising a step of reacting a polymer dissolved in an ionic liquid with a crosslinking agent while dispersing the polymer droplets in an organic solvent having low compatibility with the ionic liquid.
2) The method according to 1) above, further comprising a step of adding a medium that is compatible with the ionic liquid and does not substantially dissolve the polymer to solidify the droplets.
3) The method of 1) or 2) above, wherein the polymer is one or more selected from polysaccharides and derivatives thereof.
4) The method of 3) above, wherein the polysaccharide is one or more selected from cellulose, agarose, chitosan, dextran, pullulan, and derivatives thereof.
5) The method according to 4) above, wherein the polysaccharide contains at least cellulose or a derivative thereof.
6) The method according to 1) to 5) above, wherein the crosslinking agent is one or more selected from halohydrins, bisepoxides, polyepoxides and divinylsulfone.
7) Crosslinked polymer particles produced by the methods 1) to 6) above.
8) A chromatographic filler using the crosslinked polymer particles of 7) above.
9) A chromatographic packing material for antibody purification using the crosslinked polymer particles of 7) above.
10) The chromatographic packing material for antibody purification according to 9), wherein an affinity ligand is introduced.
11) The chromatographic packing material for antibody purification according to 10) above, wherein the affinity ligand is protein A.

  According to the present invention, since the reaction between the raw material polymer dissolved in the ionic liquid and the crosslinking agent can proceed simultaneously, the particles can be formed at the same time. As a result, it is possible to obtain crosslinked polymer particles having excellent flow rate resistance capable of feeding the mobile phase at a high flow rate. In addition, when a suitable ligand is introduced into the crosslinked polymer particles of the present invention and used as a carrier for a chromatographic filler, it is higher than when polymer particles produced by a method that does not use a crosslinking agent are used. Binding capacity is obtained. Therefore, the crosslinked polymer particles of the present invention are suitable as a carrier for a chromatographic filler for purifying biological materials such as proteins and adsorbents for antibody purification, and remarkably improve the productivity in the biological material purification step. be able to.

The graph which shows the flow resistance of a polymer particle.

In the method for producing crosslinked polymer particles of the present invention, a polymer dissolved in an ionic liquid is reacted with a crosslinking agent while droplets are dispersed in an organic solvent having low compatibility with the ionic liquid, and the formed particles are separated. It is characterized by doing.
Below, the manufacturing method of the crosslinked polymer particle of this invention and a crosslinked polymer particle are demonstrated. In the present specification, “A to B” representing a numerical range is synonymous with “A or more and B or less”, and includes A and B within the numerical range.

  In the present invention, the raw material polymer (polymer used as a raw material; hereinafter, also simply referred to as “polymer”) is not particularly limited, but it is not soluble in an organic solvent described later or is hardly soluble in an organic solvent. Those are preferred. “Slightly soluble in an organic solvent” means that the mass (g) of a polymer dissolved in 100 g of the organic solvent at room temperature (25 ° C.) is 1 g or less.

Suitable polymers include, for example, polysaccharides and water-soluble linear organic polymers such as polyvinyl alcohol, polyethyleneimine, and polyvinylamine having similar properties.
The origin of the polysaccharide is not particularly limited, and may be a natural polysaccharide or a synthetic polysaccharide. Specifically, cellulose, agarose, chitin, chitosan, dextran, pullulan, starch, guar gum, carrageenan, alginic acid, gum arabic, arabinogalactan, pectin, locust bean gum, tara gum, tamarind seed gum, psyllium seed gum, cassia gum, Examples thereof include glucomannan, xanthan gum, gellan gum, curdlan, inulin, cyclodextrin and the like, and these can be used alone or in combination.
Among these, preferable polysaccharides include cellulose, agarose, chitosan, dextran, and pullulan. Among them, cellulose and agarose are the main components from the viewpoint of high binding capacity and low nonspecific adsorption of biopolymers. Further, from the viewpoint of pressure resistance, it is preferable that cellulose is a main component.

Further, the above polymer may be a derivative in which a functional group is partially introduced as long as it dissolves in an ionic liquid.
Examples of such derivatives include those in which a part of the hydroxyl group is esterified (ester derivative) and those in which the hydroxyl group is etherified (ether derivative). Specifically, taking cellulose as an example, for example, cellulose acetate, cellulose propionate, nitrocellulose, cellulose phosphate, cellulose acetate butyrate, cellulose nitrate, methylcellulose, ethylcellulose, benzylcellulose, tritylcellulose, cyanoethylcellulose, carboxymethylcellulose Carboxyethyl cellulose, aminoethyl cellulose, oxyethyl cellulose and the like.

In the present invention, the raw material polymer is dissolved in an ionic liquid to prepare a polymer ionic liquid solution.
The ionic liquid is not particularly limited as long as the ionic liquid can uniformly dissolve the polymer. Here, “the polymer can be uniformly dissolved” includes, for example, that the polymer can be mixed with an ionic liquid in an amount of a 3% by mass solution, and dissolution can be confirmed visually. In addition, if it melt | dissolves as mentioned above, the temperature will not ask | require.

  Such an ionic liquid is composed of a cation component and an anion component, and preferably has a melting point of 200 ° C. or lower, more preferably 100 ° C. or lower, and even more preferably 50 ° C. or lower. Moreover, as a minimum of melting | fusing point, although not limited, -100 degreeC or more is preferable and -30 degreeC or more is more preferable.

  As the cation component, alkyl imidazolium cation, alkyl pyridinium cation, alkyl ammonium cation, alkyl phosphonium cation, alkyl sulfonium cation, N, N-dialkylpyrrolidinium cation, N, N-dialkylpiperidinium cation, N, N-dialkylmorphonium cation and the like can be mentioned. Of these, alkyl imidazolium cations, alkyl pyridinium cations, and alkyl ammonium cations are preferable. Here, the “alkyl” includes a linear or branched alkyl group having 1 to 12 carbon atoms, and among these, a linear alkyl group having 1 to 8 carbon atoms is preferable, and in particular, a methyl group, an ethyl group, n A linear alkyl group having 1 to 5 carbon atoms such as a -propyl group, n-butyl group, and n-pentyl group is preferred. An alkenyl group may be used instead of the alkyl group, and the alkenyl group is preferably an alkenyl group having 2 to 8 carbon atoms, and more preferably an allyl group.

  Specific examples of the cation component include N-methylimidazolium cation, N-ethylimidazolium cation, 1,3-dimethylimidazolium cation, 1,3-diethylimidazolium cation, and 1-ethyl-3-methylimidazole. 1-propyl-3-methylimidazolium cation, 1-butyl-3-methylimidazolium cation, 1-hexyl-3-methylimidazolium cation, 1,2,3-trimethylimidazolium cation and 1,2 , 3,4-tetramethylimidazolium cation, 1-allyl-3-methylimidazolium cation, N-propylpyridinium cation, N-butylpyridinium cation, 1,4-dimethylpyridinium cation, 1-butyl-4-methylpyridinium Cations and -Butyl-2,4-dimethylpyridinium cation, trimethylammonium cation, ethyldimethylammonium cation, diethylmethylammonium cation, triethylammonium cation, tetramethylammonium cation, triethylmethylammonium cation, tetraethylammonium cation, etc. 1,3-dimethylimidazolium cation, 1,3-diethylimidazolium cation, 1-ethyl-3-methylimidazolium cation, 1-propyl-3-methylimidazolium cation, 1-butyl-3-methylimidazolium Dialkyl imidazolium cations such as cation and 1-hexyl-3-methyl imidazolium cation are more preferable, and 1-ethyl-3-methyl imidazole is more preferable. Umukachion, still more preferably 1-butyl-3-methylimidazolium cation.

Examples of the anion component include halide ions (Cl ⁻ , Br ⁻ , I ^{− and the} like), carboxylate anions (for example, C ₂ H ₅ CO ₂ ⁻ , CH ₃ CO ₂ ⁻ , HCO ₂ ^{− and the} like having 1 to 3 carbon atoms in total). ), Pseudohalide ions (for example, CN ⁻ , SCN ⁻ , OCN ⁻ , ONC ⁻ , N ₃ ⁻ etc. which are monovalent and have similar properties to halides), sulfonate anions, organic sulfonate anions (methanesulfone) Acid anions, etc.), phosphate anions (ethyl phosphate anions, methyl phosphate anions, hexafluorophosphate anions, etc.), borate anions (tetrafluoroborate anions, etc.), perchlorate anions, etc., halide ions, Carboxylic acid anions are preferred.

  The ionic liquid of the present invention is preferably an alkyl imidazolium salt or a dialkyl imidazolium salt from the viewpoint of the solubility, melting point, viscosity, etc. of the polymer, and more preferably an alkyl imidazolium acetate or an alkyl imidazole. Examples of the lithium chloride include dialkylimidazolium acetate and dialkylimidazolium chloride, and more preferably 1-ethyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazolium chloride.

The dissolution of the raw material polymer in the ionic liquid varies depending on the type of polysaccharide and ionic liquid used, but is usually 10 to 250 ° C., preferably 25 to 120 ° C., for about 1 to 24 hours with stirring. It is preferred to do so.
Here, the ionic liquid has a total polymer concentration in the solution that is difficult to form droplets due to an increase in the viscosity of the solution, and suppresses frequent occurrence of microparticles during droplet formation due to a decrease in the concentration of the polymer. It is preferable to use 3 to 50% by mass, preferably 6 to 30% by mass.
In addition, the pore diameter of the particle | grains obtained changes with the melt | dissolution density | concentration to the ionic liquid of raw material polymer | macromolecule. That is, there is a tendency for the pore diameter of the particles to decrease as the polymer concentration increases. Therefore, particles having an arbitrary pore size can be obtained by adjusting the total polymer concentration within the above range. The pore diameter of the crosslinked polymer particles of the present invention is not particularly limited, but is generally about several nm to several μm when used as a packing material for chromatography.

The polymer ionic liquid solution prepared as described above is added to an organic solvent having low compatibility with the ionic liquid (hereinafter, also simply referred to as “organic solvent”), and suspended and dispersed. At this time, the polymer and the crosslinking agent are reacted.
Here, “an organic solvent having low compatibility with the ionic liquid” means that an interface is formed between the ionic liquid and the organic solvent when the ionic liquid and the organic solvent are mixed at a volume ratio of 1: 1. Means organic solvent.

  The organic solvent used here is a dispersion medium, but the type thereof is not limited as long as it has low compatibility with the ionic liquid. For example, n-hexane, n-heptane, liquid paraffin, etc. Examples include aliphatic hydrocarbons, cycloaliphatic hydrocarbons such as cyclohexane, methylcyclohexane, cycloheptane, and methylcycloheptane, and aromatic hydrocarbons such as benzene, toluene, xylene, dichlorobenzene, and the like. It may be used in a mixture of two or more. Aromatic hydrocarbons are preferable, and toluene is more preferable.

  The amount of the organic solvent used may be appropriately determined in consideration of the particle diameter of the polymer particles and the like, but is preferably 50 to 2000 parts by mass, more preferably 50 to 500 parts by mass with respect to 100 parts by mass of the ionic liquid. It is.

  The crosslinking agent used in this reaction is not particularly limited as long as it contains a functional group that reacts with the raw material polymer. For example, halohydrins such as epichlorohydrin, ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether Bisepoxides such as polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyepoxides such as glycerol polyglycidyl ether, diglycerol polyglycidyl ether, sorbitol polyglycidyl ether, trimethylolpropane polyglycidyl ether And divinyl sulfone. These crosslinking agents may be used alone or as a mixture of two or more.

  The amount of the crosslinking agent used is 0.5 to 500% by mass, preferably 1 to 200% by mass, based on the polymer, from the viewpoint of preventing particle formation and improving particle performance.

  Such a crosslinking agent is not limited in its addition method as long as it does not interfere with the reaction with the polymer. For example, it may be added in advance to a polymer ionic liquid solution, or it may be added to the polymer ionic liquid. You may add, after mixing a solution and an organic solvent. Moreover, in order to accelerate | stimulate progress of reaction, you may add a catalyst with a crosslinking agent. There is no restriction | limiting in particular as a catalyst, A general organic acid, an inorganic acid, an organic base, and an inorganic base can be used.

  In the present invention, the crosslinking reaction is performed while dispersing a polymer ionic liquid solution in an organic solvent. That is, in the method of the present invention, the cross-linking between the polymers proceeds in the droplets, and at the same time, the particles are formed. Accordingly, the conditions for the crosslinking formation reaction are not limited as long as they do not hinder droplet dispersion, but the temperature at which the polymer does not decompose and precipitate and the crosslinking reaction proceeds, for example, room temperature to 110 ° C., preferably 50 to It can be carried out by mixing and stirring at 100 ° C. for approximately 1 to 48 hours, preferably 4 to 24 hours.

  As a method of dispersion (droplet formation), a known means such as a stirrer such as a static mixer can be used. The diameter of the droplet can be controlled by the number of rotations during stirring, and the particle diameter of the resulting particles can be controlled. That is, the larger the number of rotations, the smaller the diameter of the formed droplets. Therefore, the rotation speed of the mixer may be adjusted as appropriate according to the target particle diameter.

  In order to increase the dispersion efficiency, it is preferable to add a surfactant or a lipophilic polymer to at least one of the ionic liquid and the organic solvent. Here, the surfactant used is an organic compound having a function of reducing the surface tension and having a hydrophobic site and a hydrophilic site in the molecule, and stabilizes the droplet state of the polymer ionic liquid solution. If it can disperse | distribute with keeping, it will not be specifically limited.

As the surfactant, known anionic surfactants such as known fatty acid sodium, fatty acid potassium, sodium alkylbenzene sulfonate, sodium alkyl sulfate ester, sodium alkyl ether sulfate ester, sodium alpha olefin sulfonate, sodium alkyl sulfonate;
Amphoteric surfactants such as sodium alkylamino fatty acids, alkylbetaines, alkylamine oxides;
Sucrose fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ester, fatty acid alkanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene propylene alkyl ether, polyoxyethylene propylene fatty acid Nonionic surfactants such as esters;
Etc.
Of these, nonionic surfactants are preferable, and sorbitan monooleate (SPAN 80) and sorbitan trioleate (SPAN 85) are particularly preferable.

  The lipophilic polymer is not particularly limited as long as it can be dissolved in an organic solvent to increase the viscosity and stabilize the droplet state of the ionic liquid solution of the polymer. It is preferable to use an oily polymer, for example, a polysaccharide derivative such as a cellulose derivative, a lipophilic vinyl derivative, a lipophilic acrylic acid polymer or the like.

Examples of the cellulose derivative include methyl cellulose, ethyl cellulose, cellulose acetate, carboxymethyl cellulose, and hydroxyethyl cellulose. Examples of the lipophilic vinyl derivative include polyvinyl acetate and polyvinyl chloride. Examples of the lipophilic acrylic polymer include And ethyl acrylate / methyl methacrylate / methacrylated trimethylammonium ethyl copolymer, methyl methacrylate / ethyl acrylate copolymer, and the like.
Of these, cellulose derivatives are preferable, and ethyl cellulose is particularly preferable.
Only one kind of the lipophilic polymer can be used, but two or more kinds can be used in combination.

The lipophilic polymer preferably has a weight average molecular weight of 3,000 to 5,000,000 in consideration of the stability of the droplets and the viscosity of the dispersion medium.
In addition, a weight average molecular weight means the polystyrene conversion weight average molecular weight by gel permeation chromatography (GPC).

  The amount of the surfactant or lipophilic polymer used can be appropriately selected in consideration of the dispersion performance and the properties of the ionic liquid. For example, with respect to 100 parts by mass of the organic solvent, preferably 1 part by mass to 100 parts by mass, More preferably, it is 1 mass part-25 mass parts.

  When a surfactant or a lipophilic polymer is used, the mixing order of the polymer ionic liquid solution, the organic solvent and the surfactant is not particularly limited, but the organic solvent and the surfactant or the lipophilic polymer are not limited. The mixed solution is preferably mixed with a polymer ionic liquid solution.

  Thus, cross-linking between the polymers proceeds in the process of droplet formation. When the degree of cross-linking is high, strong particles are formed in the liquid droplets, so that the cross-linked polymer particles can be separated and recovered by solid-liquid separation using a known method such as centrifugation, filtration or decantation. it can.

On the other hand, when the degree of crosslinking is low, the polymer maintains fluidity in the droplet, so that the droplet can be brought into contact with a medium that is compatible with the ionic liquid and does not substantially dissolve the polymer. It can be solidified to obtain crosslinked polymer particles.
Here, the “medium that is compatible with the ionic liquid and does not substantially dissolve the polymer” is uniformly mixed when mixed with the ionic liquid at a volume ratio of 1: 1 at room temperature, and the medium at room temperature. It means that the mass of the polymer dissolved in 100 g is 1 g or less. Examples of such a medium include one or more polar solvents such as water, methanol, and ethanol.
The medium may be added to the above-described droplet dispersion, but a method capable of coagulating the crosslinked polymer while maintaining the droplet shape is preferable. Specifically, a dispersion of the medium is prepared by mixing and stirring the organic solvent having low compatibility with the ionic liquid, the above-mentioned medium such as water, methanol, and ethanol, and if necessary, the surfactant or the lipophilic polymer. Thereafter, a method of contacting the droplet dispersion can be mentioned. The organic solvent at this time is preferably the same type as the dispersion medium of the polymer solution.

  The amount of the medium used is preferably 20 to 2,000 parts by mass, more preferably 50 to 200 parts by mass with respect to 100 parts by mass of the ionic liquid.

  Further, the contact between the liquid droplet dispersion and the medium is preferably performed at 0 to 100 ° C., preferably 25 to 80 ° C.

The crosslinked polymer particles thus prepared can be recovered by solid-liquid separation by a known method such as centrifugation, filtration, or decantation. The recovered particles are purified by washing with a solvent compatible with the ionic liquid and the organic solvent, such as ethanol, isopropanol, butanol, etc., followed by washing with water.
The volume average particle size of the crosslinked polymer particles obtained by the method of the present invention is such that when used as a chromatographic support, the pressure increases under a high flow rate and the binding capacity of a substance to be purified such as protein decreases. Considering it, it is preferably 20 to 300 μm, more preferably 30 to 150 μm.

  Thus, according to the method of the present invention, since the formation of cross-linking proceeds in the process of dispersing the polymer droplets, it is possible to produce highly rigid particles in which the inside of the particles is efficiently cross-linked. As a result, it is possible to produce crosslinked polymer particles having excellent flow rate resistance capable of feeding the mobile phase at a high flow rate (Test Example 1 described later).

  The crosslinked polymer particles obtained by the method of the present invention may be further subjected to chemical crosslinking by a known method (for example, see US Pat. No. 4,973,683 and JP-A-2009-242770). By chemical crosslinking, the rigidity of the particles is further increased, the flow resistance is improved, and use at a higher flow rate is possible. The crosslinking agent is not particularly limited as long as it contains a functional group that reacts with the hydroxyl group of the raw material polymer. For example, halohydrins such as epichlorohydrin, ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol Biepoxides such as diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyepoxides such as glycerol polyglycidyl ether, diglycerol polyglycidyl ether, sorbitol polyglycidyl ether, trimethylolpropane polyglycidyl ether, divinyl And sulfone. These crosslinking agents may be used alone or as a mixture of two or more.

  The crosslinked polymer particles of the present invention can introduce arbitrary ligands, charged groups, hydrophobic groups and the like into the particles through one or more subsequent steps by a known method. That is, the crosslinked polymer particles of the present invention are used as a carrier for various chromatographic packing materials such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, chelate chromatography, and covalent bond chromatography. Can be used.

When the crosslinked polymer particles of the present invention are used as a carrier for a chromatographic filler, a higher binding capacity can be obtained as compared with the case of using polymer particles produced by a general method not using a crosslinking agent ( Test Example 2 below). Therefore, the crosslinked polymer particles of the present invention are suitable as a carrier for a chromatographic filler for purifying biological materials such as proteins, and can significantly improve the productivity in the biological material purification step.
For example, as an affinity ligand when used as a packing material for chromatography for antibody purification, an antigen or protein having high specificity for an antibody, protein A, protein G or a variant thereof, a peptide having antibody binding activity, or the like may be used. Among them, protein A having high specificity and binding ability for antibodies is preferably used. Therefore, a chromatographic filler suitable for antibody purification can be provided by immobilizing protein A by at least a part of the hydroxyl groups of the crosslinked polymer particles of the present invention by a known method.

  Hereinafter, the present invention will be described more specifically with reference to examples. Moreover, the following description shows the aspect of this invention generally, This invention is not limited by this description without a particular reason.

1. Evaluation method 1.1. Measurement of particle size distribution The particle size distribution was measured with a laser scattering diffraction particle size distribution analyzer LS 13 320 (Beckman Coulter). The optical model is Fluid R. I. Real 1.333, Sample R .; I. Real 1.54 Imaginary 0 was used.
From this, the volume average particle size of the cellulose particles and the coefficient of variation of the volume particle size (CV value: (standard deviation / volume average particle size) × 100) were determined.

1.2. Particle sedimentation volume The particles were dispersed in water, placed in a graduated container, and the particle sedimentation volume after standing for 12 hours was measured visually.

1.3. Measurement method of flow resistance The particles obtained in the following Examples and Comparative Examples were packed in a glass column having an inner diameter of 0.5 cm (manufactured by GE Healthcare, Tricorn 5/200 column) to a bed height of 20 cm. Each column was set in a chromatography system (manufactured by GE Healthcare, AKTA prime) and 25 ° C. pure water was allowed to flow, and the pressure difference between the column inlet and the column outlet at that time was defined as the column pressure.

1.4. Method for Measuring Binding Capacity The binding capacity of the particles obtained in the following Examples and Comparative Examples was evaluated by the dynamic binding capacity for human polyclonal IgG in the protein A binding state. That is, the protein A binding particles obtained in the following Examples and Comparative Examples were each packed in a glass column (GE Healthcare, Tricorn 5/200 column) having an inner diameter of 0.5 cm to a bed height of 20 cm. Each column was set on a chromatography system (GE Healthcare, AKTA prime) and equilibrated with 20 mM phosphate buffer (pH 7.4), and then 20 mM phosphate buffer (5 mg / ml) containing human polyclonal IgG (5 mg / ml). pH 7.4) was flowed at a linear flow rate of 300 cm / hour, and the dynamic binding capacity was determined from the amount of adsorbed human polyclonal IgG and the volume of filler when the human polyclonal IgG concentration in the eluate was 10% breakthrough (breakthrough) using an absorbance monitor. Asked. Further, the back pressure during the above measurement was measured.

Example 1 Production of crosslinked polymer particles (1)
In a 500 ml separable flask, add 1.86 g of cellulose powder (manufactured by Funakoshi Co., Ltd.) to 60 g of 1-ethyl-3-methylimidazolium acetate (manufactured by Aldrich), set in a hot water bath and heat to 95 ° C. for 2 hours. The cellulose solution was obtained by stirring and dissolving. In another 500 ml separable flask, 16 g of ethyl cellulose 45 (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 200 ml of toluene (manufactured by Wako Pure Chemical Industries, Ltd.), set in a hot water bath, stirred for 2 hours at 80 ° C. and dissolved to dissolve ethyl cellulose. Containing toluene was obtained. In another separable flask, add 12 g of ethylcellulose 45 to 150 ml of toluene, set in a warm water bath and stir at 80 ° C. for 2 hours to dissolve, then add 62.5 ml of 0.2 M aqueous sodium sulfate solution at 400 rpm at 80 ° C. The mixture was stirred for 30 minutes to obtain a W / O type emulsion. Next, 0.4 g of glycerol polyglycidyl ether (product name: Epiol G-100, manufactured by NOF Corporation) was added to the cellulose solution, and toluene containing ethylcellulose 45 was further added, followed by stirring at 300 rpm and 90 ° C. for 4 hours. The above W / O emulsion was added to this suspension and cooled to room temperature while stirring at 300 rpm. Thereafter, the mixture was poured into 1500 ml of ethanol and stirred, and after standing, the supernatant was removed by decantation. Further, decantation was performed twice with 1500 ml of ethanol, followed by filtration and washing with ethanol and pure water, and further sieving in a water bath to obtain particles having a sedimentation volume of 50 ml. The resulting particles have a volume average particle size of 64 μm, C.I. V. The value was 41%.

Example 2
25 ml (sedimentation volume) of the particles obtained in Example 1 were suspended in 25 ml of pure water, and 40 ml of a 32 wt% aqueous sodium sulfate solution was further added. Under stirring, 1 ml of 45% aqueous sodium hydroxide solution and 200 mg of NaBH ₄ were added, and the temperature was raised to 50 ° C. While stirring at 50 ° C., 1.5 ml of 45 wt% sodium hydroxide aqueous solution and 1.2 ml of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.) were added 12 times in total every 12 hours, and stirring was further continued for 12 hours. did. Thereafter, the suspension was cooled, filtered and washed in the order of pure water, ethanol, and pure water, and further sieved in a water bath to obtain particles having a sedimentation volume of 25 ml. The obtained particles have a volume average particle diameter of 62 μm and C.I. V. The value was 40%.

Example 3
5 ml (sedimentation volume) of the particles obtained in Example 2 were suction dried, and water was added to make a total suspension of 7 ml. To the above suspension, 0.8 ml of 5M aqueous sodium hydroxide solution, 24 mg of NaBH ₄ and ₄ ml of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.) were added and shaken at 25 ° C. for 8 hours. Thereafter, the reaction solution was filtered and washed in the order of pure water, ethanol and pure water to obtain particles having a sedimentation volume of 5 ml.

Example 4
5 ml (sedimentation volume) of the particles obtained in Example 3 were suspended in 45 ml of 1.5 M sodium sulfate, 0.1 M phosphate buffer (pH 6.8) containing 102 mg of protein A (product name: rPA50, manufactured by RepliGen). It became cloudy. The suspension was shaken at 25 ° C. for 24 hours and then centrifuged to remove the supernatant. To the remaining particles, 45 ml of 1 M thioglycerol and 0.5 M aqueous sodium sulfate solution (pH 8.3) was added and shaken at 25 ° C. for 4 hours, then 0.1 M citrate buffer (pH 3.2), 0.1 M hydroxylated The resultant was washed successively with an aqueous sodium solution and PBS (−) to obtain particles having a sedimentation volume of 5 ml.

Example 5
Particles were produced according to the method of Example 1 except that a mixture of two types of polysaccharides, cellulose and pullulan, was used as a raw material. That is, in a 500 ml separable flask, 60 g of 1-ethyl-3-methylimidazolium acetate (manufactured by Aldrich), 3.86 g of cellulose powder (manufactured by Funakoshi Co., Ltd.) and 0.39 g of pullulan (manufactured by Hayashibara Biochemical Research Institute) Was set in a warm water bath, heated to 95 ° C., stirred for 2 hours and dissolved to obtain a mixture of cellulose and pullulan. In another 500 ml separable flask, 16 g of ethyl cellulose 45 (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 200 ml of toluene (manufactured by Wako Pure Chemical Industries, Ltd.), set in a hot water bath, stirred for 2 hours at 80 ° C. and dissolved to dissolve ethyl cellulose. Containing toluene was obtained. In another separable flask, add 12 g of ethylcellulose 45 to 150 ml of toluene, set in a warm water bath and stir at 80 ° C. for 2 hours to dissolve, then add 62.5 ml of 0.2 M aqueous sodium sulfate solution at 400 rpm at 80 ° C. The mixture was stirred for 30 minutes to obtain a W / O type emulsion. Next, 0.4 g of glycerol polyglycidyl ether (product name: Epiol G-100, NOF Corporation) is added to the solution of the mixture of cellulose and pullulan, and the toluene containing ethyl cellulose is further added, followed by stirring at 270 rpm and 90 ° C. for 4 hours. did. The above W / O emulsion was added to this suspension and cooled to room temperature while stirring at 270 rpm. Thereafter, the mixture was poured into 1500 ml of ethanol and stirred, and after standing, the supernatant was removed by decantation. Further, decantation was performed twice with 1500 ml of ethanol, followed by filtration and washing with ethanol and pure water, and further sieving in a water bath to obtain particles having a sedimentation volume of 50 ml. The obtained particles have a volume average particle size of 65 μm, C.I. V. The value was 44%.

Example 6
Using the particles obtained in Example 5, particles having a water sedimentation volume of 25 ml were obtained by the same method as shown in Example 2. The obtained particles have a volume average particle size of 65 μm, C.I. V. The value was 35%.

Example 7
Using the particles obtained in Example 6, particles having a sedimentation volume of 5 ml were obtained by the same method as the method shown in Example 3.

Example 8
Using the particles obtained in Example 7, particles having a sedimentation volume of 5 ml were obtained in the same manner as in Example 4.

Comparative Example 1
J. et al. Chromatogr. Cellulose particles were produced according to the method described in A 1217 (2010), 1298-1304 (Non-patent Document 1). That is, in a 500 ml separable flask, 4.17 g of cellulose powder (manufactured by Funakoshi Co., Ltd.) was added to 60 g of 1-butyl-3-methylimidazolium chloride (manufactured by Aldrich), set in a hot water bath, and heated to 95 ° C. It stirred for 2 hours and melt | dissolved and the cellulose solution was obtained. In another 500 ml separable flask, 9 ml of Span 85 (molecular weight 957) (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 200 ml of mineral oil (heavy) (manufactured by Aldrich), set in a hot water bath and stirred at 80 ° C. for 2 hours. It melt | dissolved and the span85 containing mineral oil (heavy) was obtained. In another separable flask, add 6.75 ml of Span85 to 150 ml of mineral oil (heavy), set in a warm water bath and stir at 80 ° C. for 2 hours to dissolve, then add 62.5 ml of 0.2M aqueous sodium sulfate solution. Stirring was performed at 400 rpm and 80 ° C. for 30 minutes to obtain a W / O type emulsion. Next, the span 85-containing mineral oil (heavy) was added to the cellulose solution and stirred at 800 rpm and 100 ° C. for 10 minutes. The W / O emulsion was added to this suspension and cooled to room temperature while stirring at 800 rpm. Thereafter, the mixture was poured into 1500 ml of ethanol and stirred, and after standing, the supernatant was removed by decantation. Further, decantation was performed twice with 1500 ml of ethanol, followed by filtration and washing with ethanol and pure water, and further sieving in a water bath to obtain particles having a water sedimentation volume of 10 ml. This operation was repeated several times to obtain the required amount of particles. The obtained particles have a volume average particle size of 66 μm, C.I. V. The value was 48%.

Comparative Example 2
Using the particles obtained in Comparative Example 1, particles having a water sedimentation volume of 25 ml were obtained by the same method as shown in Example 2. The obtained particles have a volume average particle size of 65 μm, C.I. V. The value was 47%.

Comparative Example 3
Using the particles obtained in Comparative Example 2, particles having a sedimentation volume of 5 ml were obtained by the same method as shown in Example 3.

Comparative Example 4
Using the particles obtained in Comparative Example 3, particles having a sedimentation volume of 5 ml were obtained by the same method as that shown in Example 4.

Comparative Example 5
Particles were produced according to the method of Example 1 except that the addition of glycerol polyglycidyl ether, a crosslinking agent, was omitted. That is, 3.86 g of cellulose powder (Funakoshi Co., Ltd.) was added to 60 g of 1-ethyl-3-methylimidazolium acetate (Aldrich Co.) in a 500 ml separable flask, set in a hot water bath and heated to 95 ° C. It stirred for 2 hours and melt | dissolved and the cellulose solution was obtained. In another 500 ml separable flask, 16 g of ethyl cellulose 45 (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 200 ml of toluene (manufactured by Wako Pure Chemical Industries, Ltd.), set in a hot water bath, stirred for 2 hours at 80 ° C. and dissolved to dissolve ethyl cellulose. Containing toluene was obtained. In another separable flask, add 12 g of ethylcellulose 45 to 150 ml of toluene, set in a warm water bath and stir at 80 ° C. for 2 hours to dissolve, then add 62.5 ml of 0.2 M aqueous sodium sulfate solution at 400 rpm at 80 ° C. The mixture was stirred for 30 minutes to obtain a W / O type emulsion. Next, the ethylcellulose-containing toluene was added to the cellulose solution and stirred at 300 rpm and 90 ° C. for 10 minutes. The above W / O emulsion was added to this suspension and cooled to room temperature while stirring at 300 rpm. Thereafter, the mixture was poured into 1500 ml of ethanol and stirred, and after standing, the supernatant was removed by decantation. Further, decantation was performed twice with 1500 ml of ethanol, followed by filtration and washing with ethanol and pure water, and further sieving in a water bath to obtain particles having a water sedimentation volume of 50 ml. The obtained particles have a volume average particle size of 67 μm and C.I. V. The value was 36%.

Comparative Example 6
Using the particles obtained in Comparative Example 5, particles having a water sedimentation volume of 25 ml were obtained by the same method as shown in Example 2. The obtained particles have a volume average particle diameter of 72 μm, C.I. V. The value was 34%.

Comparative Example 7
Using the particles obtained in Comparative Example 6, particles having a sedimentation volume of 5 ml were obtained by the same method as that shown in Example 3.

Comparative Example 8
Using the particles obtained in Comparative Example 7, particles having a sedimentation volume of 5 ml were obtained by the same method as shown in Example 4.

Test Example 1 Comparison of flow resistance The flow resistance of the particles of Example 2, Example 6, Comparative Example 2, and Comparative Example 6 was evaluated according to the method described in Section 1.3. The results are shown in Table 1 and FIG. The particles obtained in Example 2 and Example 6 have a lower back pressure at a constant linear flow velocity than the particles obtained in the Comparative Example, and the liquid is fed at a large linear flow rate at which consolidation occurs in the Comparative Example particles. Was possible. That is, the particles of Example 2 and Example 6 exhibited better flow resistance than the particles of the comparative example.

Test Example 2 Comparison of Binding Capacity and Flow Rate Resistance According to the method described in Section 1.4, the dynamic binding capacity of the particles of Example 4, Example 8, Comparative Example 4, and Comparative Example 8 to human polyclonal IgG was evaluated. did. The results are shown in Table 2. The particles obtained in Example 4 and Example 8 had a larger IgG binding capacity per volume and lower back pressure than the particles obtained in the comparative example.

Claims (11)

  A method for producing crosslinked polymer particles, comprising a step of reacting a polymer dissolved in an ionic liquid with a crosslinking agent while dispersing the droplets in an organic solvent having low compatibility with the ionic liquid.   The method according to claim 1, further comprising the step of adding a medium that is compatible with the ionic liquid and does not substantially dissolve the polymer to solidify the droplets.   The method according to claim 1 or 2, wherein the polymer is one or more selected from polysaccharides and derivatives thereof.   The method according to claim 3, wherein the polysaccharide is one or more selected from cellulose, agarose, chitosan, dextran, pullulan, and derivatives thereof.   The method according to claim 4, wherein the polysaccharide comprises at least cellulose or a derivative thereof.   The method according to any one of claims 1 to 5, wherein the crosslinking agent is one or more selected from halohydrins, bisepoxides, polyepoxides and divinylsulfone.   The crosslinked polymer particle manufactured by the method of any one of Claims 1-6.   A packing material for chromatography using the crosslinked polymer particles according to claim 7.   A packing material for chromatography for antibody purification using the crosslinked polymer particles according to claim 7.   The chromatography packing material for antibody purification according to claim 9, wherein an affinity ligand is introduced.   The packing material for chromatography for antibody purification according to claim 10, wherein the affinity ligand is protein A.

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