Filler and column for liquid chromatography

Abstract

The purpose of the present disclosure is to provide a filler for liquid chromatography that reduces electrostatic interaction between a polymer compound such as a protein, a filler, and silanol of the filler, and has excellent separation characteristics for a low-molecular-weight compound having a molecular weight equal to or less than that of a modifier.　According to the present disclosure, there is used a filler for liquid chromatography comprising a carrier and a modifier bound to the carrier, wherein the modifier has a structure represented by formula (1) (the symbols in the formula are as set forth in the description).

Description

Packing materials and columns for liquid chromatography

[0001] The present disclosure relates to a packing material for liquid chromatography. The present disclosure also relates to a column packed with the packing material.

[0002] Size exclusion chromatography (SEC) is a sample separation technique that is used in various fields, including life science and pharmaceutical science. Examples of stationary phases used in SEC include silica gel and its surface modified with various atomic groups, as described in Non-Patent Document 1. However, secondary interactions such as electrostatic adsorption due to residual silanol on the silica gel surface and hydrophobic adsorption due to surface modifiers can occur, resulting in the retention of samples such as antibodies, for which there has been increasing demand in recent years, on the stationary phase.

[0003] Patent Literature 1 discloses that the presence of hydroxy-terminated polyethylene glycol (PEG) on the surface of the stationary phase reduces hydrophobic secondary interactions and electrostatic interactions between proteins and the hydrophobic moieties or silanols of the stationary phase. However, the molecular weight of the modifier is large, and substances with molecular weights smaller than this have elution positions close to each other, which is problematic in that small molecules cannot be separated, and improvements have been sought.

[0004] International Publication No. 2022 / 061039

[0005] “Modern Size-Exclusion Liquid Chromatography: Practice of Gel Filtration Chromatography (2nd edition)” by Andre Striegel. edition)” Wiley Publishing 2009

[0006] An object of the present disclosure is to provide a packing material for liquid chromatography that reduces electrostatic interactions between high molecular weight compounds such as proteins and silanols of the packing material and has good separation properties for low molecular weight compounds with molecular weights equal to or smaller than that of the modifier.

[0007] Another object of the present disclosure is to provide a column packed with the above-mentioned packing material for liquid chromatography.

[0008] The inventors of the present disclosure have conducted extensive research to solve the above problems, and as a result have discovered the following packing material for liquid chromatography, leading to the completion of the present disclosure.

[0009] That is, one aspect of the present disclosure is as follows: [1] A packing material for liquid chromatography comprising a support and a modifier bonded to the support, wherein the modifier has a structure represented by the following formula (1): In formula (1), X represents a hydrogen atom, a methyl group, or an ethyl group, Q represents a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group, m represents 1, 2, or 3, n represents an integer of 1 to 50, and the wavy line represents a bonding site between the modifying agent and the support. [2] The filler according to [1] above, wherein Q represents a urethane bond or an amide bond in formula (1). [3] The filler according to [1] or [2] above, wherein X represents a hydrogen atom or a methyl group, Q represents a urethane bond, and m represents 3 in formula (1). [4] The filler according to any one of [1] to [3] above, wherein the support is a porous support. [5] The filler according to any one of [1] to [4] above, wherein the support is one or more particles selected from the group consisting of silica particles, alumina particles, titania particles, and zirconia particles. [6] The filler according to any one of [1] to [5], wherein the average particle diameter (D50) of the support is 1 μm or more and 100 μm or less. [7] The filler according to any one of [4] to [6], wherein the average pore diameter of the support is 0.5 nm or more and 200 nm or less. [8] The filler according to any one of [4] to [6], wherein the specific surface area of ​​the support is 10 m ２ / g or more 1000m ２ The packing material according to any one of [4] to [7], wherein the solubility is 1 / g or less. [9] A column packed with the packing material for liquid chromatography according to any one of [1] to [8].

[0010] The present disclosure makes it possible to provide a packing material for liquid chromatography that reduces electrostatic interactions between high molecular weight compounds such as proteins and silanols of the packing material and has good separation properties for low molecular weight compounds with molecular weights equal to or smaller than that of the modifier.

[0011] In the present disclosure, the molecular weight of the polymer compound is not particularly limited, and may be, for example, more than 600, 700 or more, 800 or more, 900 or more, 1,000 or more, 2,000 or more, 3,000 or more, 4,000 or more, 5,000 or more, 6,000 or more, 7,000 or more, 8,000 or more, 9,000 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, or 50,000 or more, and may be 100,000,000 or less, 10,000,000 or less, 1,000,000 or less, 100,000 or less, 10,000 or less, or 1,000 or less. Furthermore, the molecular weight of the low molecular weight compound is not particularly limited and may be, for example, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 150 or less, or may be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more.

[0012] Fig. 1 is a diagram showing a chromatogram obtained by separating a low molecular weight compound sample using a packing material according to an example of the present disclosure; Fig. 2 is a diagram showing a chromatogram obtained by separating a low molecular weight compound sample using a packing material according to a comparative example; Fig. 3 is a diagram showing a chromatogram obtained by separating a protein using a packing material according to the present disclosure; Fig. 4 is a diagram showing a chromatogram obtained by separating a protein using a packing material according to a comparative example.

[0013] The filler of the present disclosure will be described below with reference to an example embodiment. The present disclosure includes any combination of the configurations and parameters disclosed herein, and also any combination of the upper and lower limits of the values ​​disclosed herein.

[0014] The present embodiment is a packing material for liquid chromatography comprising a support and a modifier bonded to the support, wherein the modifier has a structure represented by the following formula (1): In formula (1), X represents a hydrogen atom, a methyl group, or an ethyl group; Q represents a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group; m represents 1, 2, or 3; n represents an integer of 1 or more and 50 or less; and the wavy line represents a bonding site between the modifying agent and the carrier.

[0015] In formula (1), X represents a hydrogen atom, a methyl group, or an ethyl group. X is preferably a hydrogen atom or a methyl group, since the filler is less likely to cause hydrophobic interactions and has excellent hydrophilicity.

[0016] In formula (1), Q represents a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group. The urethane bond is a bond represented by -NH-CO-O-, and the directionality of the bond may be either. The amide bond is a bond represented by -NH-CO-, and the directionality of the bond may be either. The 2-oxopropane-1,3-diyl group is a bond represented by -CH ２ -CO-CH ２ - is a group represented by the formula:

[0017] Of these, Q is preferably an amide bond or a urethane bond, and more preferably a urethane bond, because the presence of an amide bond on the carrier surface is effective in reducing the electrostatic interaction effect of acidic silanols.

[0018] In formula (1), m represents 1, 2, or 3. m is preferably 2 or 3, and more preferably 3, in terms of excellent performance in selectively separating low-molecular-weight compounds and high-molecular-weight compounds through hydrophobic interaction.

[0019] In formula (1), n ​​represents an integer of 1 or more and 50 or less. The value of n can be selected according to the molecular weight of the sample to be separated by chromatography. When the sample is a polymer compound, n is preferably an integer of 8 or more and 22 or less, and more preferably an integer of 8 or more and 10 or less, in terms of excellent shielding effect of blocking access to surface silanols. When the compound is a low molecular weight compound, n is preferably an integer of 40 or more and 50 or less.

[0020] The modifying agent represented by formula (1) preferably has a structure represented by formula (2) or (3) in terms of excellent hydrophilicity and steric exclusion effect due to hydration of polyethylene glycol (PEG). Here, the wavy line represents the binding site between the modifier and the carrier.

[0021] When X in formula (1) is a hydrogen atom, the hydroxyl-terminated PEG is bifunctional, and therefore the carrier of this embodiment may contain a crosslinked PEG-containing group represented by formula (4). Here, the wavy line represents the binding site between the modifier and the carrier.

[0022] The average particle diameter (hereinafter also referred to as "D50") of the carrier of this embodiment is preferably 1 μm or more and 100 μm or less. For example, the average particle diameter of the carrier of the present disclosure may be 1.0 μm or more, 1.1 μm or more, 1.2 μm or more, 1.3 μm or more, 1.4 μm or more, 1.5 μm or more, 1.6 μm or more, or 1.7 μm or more, or 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 9.0 μm or less, 8.0 μm or less, 7.0 μm or less, 6.0 μm or less, 5.0 μm or less, 4.0 μm or less, 3.0 μm or less, 2.0 μm or less, 1.9 μm or less, or 1.8 μm or less. Therefore, for example, when the average particle size of the carrier of the present disclosure is 1.0 μm or more and 1.8 μm or less, 1.0 μm or more and 1.9 μm or less, 1.0 μm or more and 2.0 μm or less, 1.0 μm or more and 3.0 μm or less, 1.0 μm or more and 4.0 μm or less, 1.0 μm or more and 5.0 μm or less, 1.0 μm or more and 10 μm or less, 1.0 μm or more and 50 μm or less, 1.5 μm or more and 1.8 μm or less, 1.5 μm or more and 1.9 μm or less, 1.5 μm or more and 2.0 μm or less, 1.5 μm or more and ... or more and 3.0 μm or less, 1.5 μm or more and 4.0 μm or less, 1.5 μm or more and 5.0 μm or less, 1.5 μm or more and 10 μm or less, 1.5 μm or more and 50 μm or less, 1.7 μm or more and 1.8 μm or less, 1.7 μm or more and 1.9 μm or more, 1.7 μm or more and 2.0 μm or less, 1.7 μm or more and 3.0 μm or less, 1.7 μm or more and 4.0 μm or less, 1.7 μm or more and 5.0 μm or less, 1.7 μm or more and 10 μm or less, or 1.7 μm or more and 50 μm or less.

[0023] Since the particle size of the filler according to this embodiment corresponds to the particle size of the carrier used, a carrier having a desired average particle size can be selected. Generally, in an embodiment in which a modifying agent is bonded to a carrier, the average particle size before and after bonding does not change beyond a margin of error, so the average particle size of the filler can be considered to be the same as the average particle size of the carrier used.

[0024] Therefore, the average particle size of the filler according to this embodiment is preferably 1 μm or more and 100 μm or less. For example, the average particle size of the filler according to the present disclosure may be 1.0 μm or more, 1.1 μm or more, 1.2 μm or more, 1.3 μm or more, 1.4 μm or more, 1.5 μm or more, 1.6 μm or more, or 1.7 μm or more, or may be 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 9.0 μm or less, 8.0 μm or less, 7.0 μm or less, 6.0 μm or less, 5.0 μm or less, 4.0 μm or less, 3.0 μm or less, 2.0 μm or less, 1.9 μm or less, or 1.8 μm or less. These upper and lower limit values ​​may be in any combination. Therefore, for example, when the average particle size of the filler of the present disclosure is 1.0 μm or more and 1.8 μm or less, 1.0 μm or more and 1.9 μm or less, 1.0 μm or more and 2.0 μm or less, 1.0 μm or more and 3.0 μm or less, 1.0 μm or more and 4.0 μm or less, 1.0 μm or more and 5.0 μm or less, 1.0 μm or more and 10 μm or less, 1.0 μm or more and 50 μm or less, 1.5 μm or more and 1.8 μm or less, 1.5 μm or more and 1.9 μm or less, 1.5 μm or more and 2.0 μm or less, 1.5 μm or more and ... or more and 3.0 μm or less, 1.5 μm or more and 4.0 μm or less, 1.5 μm or more and 5.0 μm or less, 1.5 μm or more and 10 μm or less, 1.5 μm or more and 50 μm or less, 1.7 μm or more and 1.8 μm or less, 1.7 μm or more and 1.9 μm or more, 1.7 μm or more and 2.0 μm or less, 1.7 μm or more and 3.0 μm or less, 1.7 μm or more and 4.0 μm or less, 1.7 μm or more and 5.0 μm or less, 1.7 μm or more and 10 μm or less, or 1.7 μm or more and 50 μm or less.

[0025] The average particle size of this embodiment can be determined by the Coulter method using a general particle size distribution measuring device (for example, product name: Multisizer 3, manufactured by Beckman Coulter). The Coulter method is generally recognized as an electrical resistance method. The measurement conditions can be exemplified as follows.

[0026] <Measurement sample> Silica gel (measurement sample): approx. 0.0010 g

[0027] <Pretreatment> Electrolyte: ISOTON-II (manufactured by BECKMAN COULTER) Ultrasonic disperser: BRANSONIC M2800-J Frequency: 40 kHz Treatment time: 1 minute

[0028] <Measuring Average Particle Diameter> The pretreated silica gel (measurement sample) is measured using a particle size distribution analyzer (Multisizer 3, manufactured by BECKMAN COULTER) under conditions of an aperture diameter of 50 μm and a particle count of 50,000, and the volume average particle diameter can be determined from the volume particle size distribution of the sample particles.

[0029] The carrier of the present embodiment is not particularly limited, but examples thereof include inorganic particles and resin particles, and more specifically, examples thereof include silica particles, alumina particles, titania particles, zirconia particles, magnesia particles, hydroxyapatite particles, silicone particles, polyacrylic acid particles, polymethacrylic acid particles, nylon particles, and polyamide particles.

[0030] The carrier is preferably one or more particles selected from the group consisting of silica particles, alumina particles, titania particles, and zirconia particles, and more preferably silica particles, because of their excellent mechanical strength.

[0031] The carrier of this embodiment may be porous or non-porous, but is preferably a porous particle because it excels in molecular weight distribution-dependent separation as a packing for size exclusion chromatography. That is, the particles are more preferably one or more particles selected from the group consisting of porous silica particles, porous alumina particles, porous titania particles, and porous zirconia particles, and more preferably porous silica particles.

[0032] Examples of porous particles include silica monoliths, alumina monoliths, silica-alumina monoliths, polymethylsilsesquioxane monoliths, titanium oxide monoliths, polyacrylamide monoliths, and polymethacrylic acid ester monoliths. Monoliths are generally recognized as porous bodies.

[0033] The average pore diameter of the support of this embodiment is preferably 0.5 nm or more and 200 nm or less. For example, the average pore diameter of the support may be 0.5 nm or more, 1.0 nm or more, 2.0 nm or more, 3.0 nm or more, 4.0 nm or more, 5.0 nm or more, 6.0 nm or more, 7.0 nm or more, 8.0 nm or more, 9.0 nm or more, 10.0 nm or more, 15 nm or more, 16 nm or more, 17 nm or more, 18 nm or more, 19 nm or more, or 20 nm or more, or may be 200 nm or less, 100 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, or 25 nm or less. Any combination of these upper and lower limits may be used. Therefore, for example, the average pore diameter of the support may be 1.0 nm or more and 25 nm or less, 1.0 nm or more and 30 nm or less, 1.0 nm or more and 40 nm or less, 1.0 nm or more and 50 nm or less, 1.0 nm or more and 100 nm or less, 1.0 nm or more and 200 nm or less, 5.0 nm or more and 25 nm or less, 5.0 nm or more and 30 nm or less, 5.0 nm or more and 40 nm or less, 5.0 nm or more and 50 nm or less, 5.0 nm or more and 100 nm or less, 5.0 nm or more and 200 nm or less, 10 nm or more and 25 nm or less, 10 nm or more and 30 nm or less, 10 nm or more and 40 nm or less, 10 nm or more and 50 nm or less, 10 nm or more and 100 nm or less, 10 nm or more and 200 nm or less, 15 nm or more and 30 nm or less, or 19 nm or more and 30 nm or less.

[0034] The specific surface area of ​​the carrier in this embodiment is 10 m ２ / g or more 1000m ２ For example, the specific surface area of ​​the carrier is preferably 10 m ２ / g or more, 20m ２ / g or more, 30m ２ / g or more, 40m ２ / g or more, 50m ２ / g or more, or 100m ２ / g or more, 150m ２ / g or more, or 200m ２ / g or more, and ２ / g or less, 900m ２ / g or less, 800m ２ / g or less, 700m ２ / g or less, 600m ２ / g or less, 500m ２ / g or less, 400m ２ / g or less, 300m ２ / g or less, or 250m ２ / g or less. Any combination of these upper and lower limits is acceptable. Therefore, for example, the specific surface area of ​​the carrier may be 10 m ２ / g or more 250m ２ / g or less, 10m ２ / g or more 300m ２ / g or less, 10m ２ / g or more 400m ２ / g or less, 10m ２ / g or more 500m ２ / g or less, 10m ２ / g or more 600m ２ / g or less, 50m ２ / g or more 250m ２ / g or less, 50m ２ / g or more 300m ２ / g or less, 50m ２ / g or more 400m ２ / g or less, 50m ２ / g or more 500m ２ / g or less, 50m ２ / g or more 600m ２ / g or less, 100m ２ / g or more 250m ２ / g or less, 100m ２ / g or more 300m ２ / g or less, 100m ２ / g or more 400m ２ / g or less, 100m ２ / g or more 500m ２ / g or less, 100m ２ / g or more 600m ２ / g or less, 200m ２ / g or more 250m ２ / g or less, 200m ２ / g or more 300m ２ / g or less.

[0035] Since the specific surface area of ​​the filler according to this embodiment corresponds to the specific surface area of ​​the carrier used, a carrier having a desired specific surface area may be selected. Generally, in an embodiment in which a modifier is bonded to a carrier, the specific surface area before and after bonding does not change beyond a margin of error, and therefore the specific surface area of ​​the filler can be considered to be the same as the specific surface area of ​​the carrier used.

[0036] Therefore, the specific surface area of ​​the filler of this embodiment is 10 m ２ / g or more 1000m ２ For example, the specific surface area of ​​the filler is preferably 10 m ２ / g or more, 20m ２ / g or more, 30m ２ / g or more, 40m ２ / g or more, 50m ２ / g or more, 100m ２ / g or more, 150m ２ / g or more, or 200m ２ / g or more, and ２ / g or less, 900m ２ / g or less, 800m ２ / g or less, 700m ２ / g or less, 600m ２ / g or less, 500m ２ / g or less, 400m ２ / g or less, 300m ２ / g or less, or 250m ２ / g or less. Any combination of these upper and lower limits may be used. Therefore, for example, the specific surface area of ​​the filler may be 10 m ２ / g or more 250m ２ / g or less, 10m ２ / g or more 300m ２ / g or less, 10m ２ / g or more 400m ２ / g or less, 10m ２ / g or more 500m ２ / g or less, 10m ２ / g or more 600m ２ / g or less, 50m ２ / g or more 250m ２ / g or less, 50m ２ / g or more 300m ２ / g or less, 50m ２ / g or more 400m ２ / g or less, 50m ２ / g or more 500m ２ / g or less, 50m ２ / g or more 600m ２ / g or less, 100m ２ / g or more 250m ２ / g or less, 100m ２ / g or more 300m ２ / g or less, 100m ２ / g or more 400m ２ / g or less, 100m ２ / g or more 500m ２ / g or less, 100m ２ / g or more 600m ２ / g or less, 200m ２ / g or more 250m ２ / g or less, 200m ２ / g or more 300m ２ / g or less.

[0037] The average pore size and specific surface area of ​​the carrier of this embodiment may be measured by a gas adsorption method using a general gas adsorption amount measuring device (product name: BELSORP MINI II, manufactured by MicrotracBEL).

[0038] The gas adsorption method is a method in which a porous sample is allowed to adsorb a gas by changing the pressure, the amount of adsorption at that time is measured, and the specific surface area, pore volume, pore distribution, etc. are calculated from an adsorption / desorption isotherm in which the relative pressure (the ratio of the pressure in the adsorption equilibrium state to the saturated vapor pressure) and the amount of adsorption are plotted. JIS Z8831-2 (mesopores and macropores) or JIS Z 8831-3 (micropores), which specify the pore size distribution and pore characteristics of powders (solids), can be applied mutatis mutandis.

[0039] The support of this embodiment can be produced by reacting the above-mentioned support with a compound represented by formula (5). In formula (5), X is a hydrogen atom, a methyl group, or an ethyl group, Q is a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group, m is 1, 2, or 3, and n is an integer of 1 or more and 50 or less. １ is OCH ３ , O.C. ２ H ５ , O.C. ３ H ７ , OCH(CH ３ ) ２ , O.C. ２ H ４ OCH ３ , OC(=O)CH ３ , N(CH ３ ) ２ , or Cl, and R ２ and R ３ are each independently H, CH ３ , C ２ H ５ , C ３ H ７ OCH ３ , O.C. ２ H ５ , O.C. ３ H ７ , OCH(CH ３ ) ２ , O.C. ２ H ４ OCH ３ , OC(=O)CH ３ , N(CH ３ ) ２ and Cl.

[0040] The solvent for bonding the modifying agent to the support is not particularly limited, and may be, for example, one or more solvents selected from the group consisting of toluene, xylene, ethylene glycol, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butyl alcohol, acetone, dichloromethane, 1,2-dichloroethane, chloroform, tetrahydrofuran, 1,4-dioxane, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, water, and an aqueous solution containing 0.001 to 5.0 mol / L of a salt.

[0041] In the aqueous solution containing 0.001 to 5.0 mol / L of salt, the type of salt may be one or more selected from the group consisting of sodium phosphate, potassium phosphate, ammonium formate, ammonium acetate, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, sodium citrate, sodium formate, and sodium acetate.

[0042] The temperature at which the modifying agent is bonded to the carrier is not particularly limited, but may be from 0° C. to 300° C., and is preferably the reflux temperature of the solvent.

[0043] The concentration of the modifying agent is preferably 0.01 mmol or more and 4.0 mmol or less per 1 g of carrier. For example, the modifying agent of the present disclosure may be 0.01 mmol or more, 0.05 mmol or more, 0.1 mmol or more, 0.5 mmol or more, or 1.0 mmol or more per 1 g of carrier, or 4.0 mmol or less, 3.0 mmol or less, 2.5 mmol or less, or 2.0 mmol or less. Any combination of these upper and lower limits may be used. Therefore, for example, the concentration of the modifying agent of the present disclosure may be 0.1 mmol or more and 3.0 mmol or less, 0.1 mmol or more and 2.5 mmol or less, 0.1 mmol or more and 2.0 mmol or less, 0.5 mmol or more and 3.0 mmol or less, 0.5 mmol or more and 2.5 mmol or less, 0.5 mmol or more and 2.0 mmol or less, 1.0 mmol or more and 3.0 mmol or less, 1.0 mmol or more and 2.5 mmol or less, or 1.0 mmol or more and 2.0 mmol or less per 1 g of carrier.

[0044] The catalyst for bonding the modifying agent to the support is not particularly limited, and may be, for example, one or more selected from the group consisting of ammonia, methylamine, ethylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyridine, aniline, pyrrole, sodium hydroxide, potassium hydroxide, hydrochloric acid, nitric acid, sulfuric acid, formic acid, acetic acid, trifluoroacetic acid, and boron trifluoride.

[0045] The compound represented by formula (5) may be a commercially available product, or may be produced by a generally known method.

[0046] The packing material of this embodiment can be used for chromatography, for example, after being packed in a column, and is preferably used for size exclusion chromatography. The retention factor (k) can be used as an index for evaluating the separation performance of the packing material of this embodiment for low molecular weight compound samples. The retention factor (k) is defined as follows: (Retention Factor) Retention factor k=(t Ｒ -t ０ ) / t ０ t Ｒ : sample elution time t ０ : Elution time of unretained sample

[0047] In this embodiment, t ０ is the elution time of uracil.

[0048] In this embodiment, a large retention factor is considered to indicate improved separation performance for low molecular weight compound samples in the packing material of this embodiment. The elution time is defined as the time when the sample is injected, and the elution time is defined as the time when a perpendicular line is dropped from the peak top of each sample in the chromatogram.

[0049] The peak area and the ratio of the peak to the total area can be used as indicators for evaluating the effect of the filler of this embodiment in reducing the electrostatic interaction between the polymer compound and the silanol of the filler. The ratio of the peak to the total area is defined as follows: (Ratio of the peak to the total area) Ratio of the peak to the total area = A１ / A ａｌｌ A １ : Peak area of ​​sample A ａｌｌ : Peak area of ​​all peaks

[0050] In this embodiment, A １ is the peak area of ​​tri-PEGylated lysozyme, which is most adsorbed by hydrogen bonding with the remaining silanol groups on the surface of the porous silica gel.

[0051] In this embodiment, a large peak area and a large ratio of the peak to the total area are evaluated as indicating an improved effect of reducing the electrostatic interaction between the polymer compound and the silanol of the filler in this embodiment.

[0052] The sample to be separated by the filler of this embodiment is not particularly limited, but examples thereof include sugars, proteins, PEGylated proteins, peptides, oligonucleotides, nucleic acids, antibodies, antibody-drug conjugates, enzymes, extracellular vesicles, adeno-associated viruses, lipid nanoparticles, liposomes, and lentiviruses.

[0053] The present disclosure will be described below with reference to examples, but the present disclosure is not limited thereto.

[0054] (Particle size distribution measurement) A volume particle size distribution curve of the sample was obtained using the HRA mode of a precision particle size distribution measuring device (product name: Multisizer 3 (aperture diameter 50 μm), manufactured by BECKMAN COULTER), and the median diameter (D50) was measured from this. Prior to the measurement, the sample was suspended in pure water and dispersed for 1 minute using an ultrasonic homogenizer as a pretreatment.

[0055] (Measurement of specific surface area) The adsorption / desorption isotherm by nitrogen adsorption was measured using a gas adsorption amount measuring device (product name: BELSORP MINI II, manufactured by MicrotracBEL) by the constant volume method, and the specific surface area a ｓ、ＢＥＴ was calculated.

[0056] (Pore diameter measurement) The adsorption / desorption isotherm by nitrogen adsorption was measured using a gas adsorption amount measurement device (product name: BELSORP MINI II, manufactured by MicrotracBEL) by a constant volume method, and the total pore volume V was calculated from the pore distribution on the desorption side by the BJH method. ｐ The average pore diameter D was calculated as follows: ｐ / a ｓ、ＢＥＴ It was calculated by:

[0057] Example 1 (1-1) Synthesis of modifying agent 2.47 g of (3-isocyanatopropyl)trimethoxysilane (CAS RN 15396-00-6, manufactured by Tokyo Chemical Industry Co., Ltd.) and 5.29 g of polyethylene glycol monomethyl ether 400 (CAS RN 9004-74-4, manufactured by Tokyo Chemical Industry Co., Ltd., average molecular weight 380 to 420) were mixed in a glass vial. The glass vial was heated to 75°C in an oil bath and maintained at this temperature for 12 hours with stirring to obtain a modifying agent.

[0058] (1-2) Binding to a support Porous silica gel (D50: 1.78 μm, average pore diameter: 20.1 nm, specific surface area: 231 m) dried overnight at 110°C in a vacuum ２ 4.00 g of PEG-400 / g and 80 mL of a 10 mmol / L aqueous sodium phosphate solution (pH 6.0) were mixed, and the mixture was irradiated with ultrasonic waves for 1 minute to disperse the porous silica gel.

[0059] Next, the modifying agent obtained in (1-1) was added in an amount of 1 mmol per 1 g of porous silica gel, and the mixture was heated to 99°C in an oil bath and maintained for 24 hours with stirring. After the silane coupling reaction was completed, the resulting powder was collected by filtration and washed five times with 100 mL of 70°C hot water to obtain the filler of this example.

[0060] The filler can be assumed to have the following structure:

[0061] Example 2 The packing material of this example was obtained in the same manner as in Example 1, except that in (1-2), the modifying agent obtained in (1-1) was added in an amount of 2 mmol per 1 g of porous silica gel.

[0062] It can be assumed that the packing material has the same structure as that of Example 1. In the case of Example 2, the amount of the modifying agent added is greater than that of Example 1, and therefore it can be assumed that the density of the modifying groups that can be generated (i.e., the modifying groups bonded to the carrier) is higher than that of Example 1.

[0063] Example 3 A filler of this example was obtained in the same manner as in Example 2, except that 2.06 g of (3-isocyanatopropyl)trimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) and 4.40 g of PEG 400 (CAS RN is 25322-68-3, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., average molecular weight 360 to 440) were used instead of polyethylene glycol monomethyl ether 400 in Example 2.

[0064] It is estimated that the filler has the following structure: Note that, since a modifier having a crosslinked structure is contained when the modifier is synthesized, it is estimated that the following structure may be generated.

[0065] Example 4 A filler of this example was obtained in the same manner as in Example 3, except that 40 mL of a 10 mmol / L aqueous solution of ammonium acetate (pH 9.5) was mixed instead of the 10 mmol / L aqueous solution of sodium phosphate (pH 6.0).

[0066] It can be assumed that the filler has the same structure as in Example 3.

[0067] Example 5 A filler of this example was obtained in the same manner as in Example 3, except that 2.90 g of (3-isocyanatopropyl)trimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) and 42.03 g of PEG2000 (CAS RN 25322-68-3, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., average molecular weight 1,800 to 2,200) were used instead of PEG400. The filler can be estimated to have the following structure. This structure can be estimated in the same manner as in Example 3.

[0068] Comparative Example 1 A chromatography column packed with diol-terminated porous silica gel (trade name: TSKgel UP-SW3000-LS, inner diameter 4.6 mm, length 15 cm, manufactured by Tosoh Corporation) was used.

[0069] Comparative Example 2 Porous silica gel (D50: 1.78 μm, average pore diameter: 20.1 nm, specific surface area: 231 m) dried overnight at 110° C. ２ 5.00 g of PEG-400 / g and 100 mL of a 10 mmol / L aqueous sodium phosphate solution (pH 6.0) were mixed, and the mixture was irradiated with ultrasonic waves for 1 minute to disperse the porous silica gel.

[0070] Next, 3-[methoxy(polyethyleneoxy)6-9propyl]trimethoxysilane (CAS RN 65994-07-2, manufactured by Gelest, teck-90) was added in an amount of 2 mmol per 1 g of porous silica gel, and the mixture was heated to 99°C in an oil bath and maintained for 24 hours with stirring. After completion of the silane coupling reaction, the resulting powder was collected by filtration and washed five times with 100 mL of 70°C hot water to obtain the filler of this comparative example.

[0071] The filler can be assumed to have the following structure:

[0072] Comparative Example 3 Porous silica gel without a modifier (D50: 1.78 μm, average pore diameter: 20.1 nm, specific surface area: 231 m ２ / g) was used as the filler in this comparative example.

[0073] Comparative Example 4 A filler for this comparative example was obtained in the same manner as in Example 3, except that 2.89 g of (3-isocyanatopropyl)trimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) and 63.02 g of PEG 4000 (CAS RN is 25322-68-3, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., average molecular weight 2,700 to 3,300) were used instead of PEG 400 in Example 3.

[0074] Comparative Example 5 In Comparative Example 4, the porous silica gel had a D50 of 3.82 μm, an average pore diameter of 111 nm, and a specific surface area of ​​28 m ２The filler of this comparative example was obtained in the same manner as in Comparative Example 4, except that a filler having a molecular weight of 1 / g was used.

[0075] The packing materials obtained in Examples 1 to 5 and Comparative Examples 2 to 5 were packed into a liquid chromatography column having an inner diameter of 4.6 mm and a length of 15 cm. These packing materials are shown in Table 1.

[0076]

[0077] [Separation of low molecular weight compound samples] Using the liquid chromatography columns obtained in Examples 1 to 5 and Comparative Examples 1 to 5, uracil (molecular weight: 112.09), benzoic acid (molecular weight: 122.12), p-aminobenzoic acid (molecular weight: 137.14), and p-hydroxybenzoic acid (molecular weight: 138.12) were separated under the following measurement conditions.

[0078] The elution time of uracil is t ０ The elution time of each sample is t Ｒ Then, the retention coefficient k is (t Ｒ -t ０ ) / t ０ The obtained chromatograms are shown in Figures 1A and 1B, and the retention coefficient k of each substance is shown in Table 2.

[0079] (Measurement conditions) Eluent: 100 mmol / L phosphate buffer (pH 5.0), 100 mmol / L sodium sulfate, and 0.05% sodium azide Flow rate: 0.35 mL / min Detector: SPD-20A (Shimadzu Corporation) Detection: UV 280 nm Temperature: 25°C Sample concentrations: 0.03 g / L uracil, 0.5 g / L benzoic acid, 0.01 g / L p-aminobenzoic acid, 0.1 g / L p-hydroxybenzoic acid Sample injection volume: 5 μL

[0080] In Figures 1A and 1B, peak 1 represents uracil, peak 2 represents benzoic acid, peak 3 represents p-aminobenzoic acid, and peak 4 represents p-hydroxybenzoic acid.

[0081] As can be seen from Table 2, the retention coefficients k of Examples 1 to 5 were greater than those of Comparative Examples 1, 2, 3, and 5 for all low molecular weight compound samples, confirming that the packing material of this embodiment is excellent at separating low molecular weight compound samples located near the permeation limit. On the other hand, in Comparative Example 2, in which the modifier did not contain a urethane bond, it was confirmed that peak 3 of p-aminobenzoic acid and peak 4 of p-hydroxybenzoic acid overlapped, making clear separation impossible. Furthermore, in Comparative Example 3, which was porous silica gel without a modifier, it was confirmed that peak 1 of uracil and peak 3 of p-aminobenzoic acid overlapped, making clear separation impossible. Furthermore, in Comparative Example 1, the retention coefficient k was smaller than in Examples 1 to 5, confirming that the separation state was not good. Furthermore, in Comparative Examples 4 and 5, it was confirmed that peak 2 of benzoic acid, peak 3 of p-aminobenzoic acid, and peak 4 of p-hydroxybenzoic acid broadened, resulting in poor separation. From this, it was found that a packing material for liquid chromatography in which, in formula (1), X is a hydrogen atom, a methyl group, or an ethyl group, Q is a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group, m is 1, 2, or 3, and n is an integer of 1 to 50 exhibits excellent separation properties for low molecular weight compounds. Furthermore, since the separation properties for low molecular weight compounds are improved when Q in formula (1) is a urethane bond to the oxygen atom, it can be understood that an amide bond is necessary on the surface of the carrier.

[0082]

[0083] [Separation of Proteins] Using the columns of Examples 1 to 5, which showed good separation ability for low molecular weight compound samples, and Comparative Examples 1 and 4, which showed no peak overlap, an attempt was made to separate PEGylated proteins, which are adsorbed by hydrogen bonding with the remaining silanol groups on the porous silica gel surface.

[0084] Lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, derived from egg white, molecular weight: 14,307) was used as an example of a protein. 15 mg of the lysozyme, 16 mg of SUNBRIGHT ME-050CS (manufactured by NOF Corp.), and 10 mL of 10 mmol / L phosphate buffer (pH 6.5) were mixed and stirred at 25°C for 2 hours to obtain PEGylated lysozyme.

[0085] The PEGylated lysozyme was separated under the following measurement conditions using the liquid chromatography columns obtained in Examples 1 to 5 and Comparative Examples 1 and 4. The chromatograms obtained are shown in Figures 2A and 2B. Table 3 also shows the peak area of ​​Peak 1, which is tri-PEGylated lysozyme, and the ratio of Peak 1 to the total area.

[0086] (Measurement conditions) Eluent: 100 mmol / L phosphate buffer (pH 6.7), 100 mmol / L sodium sulfate, and 0.05% sodium azide Flow rate: 0.35 mL / min Detector: SPD-20A (Shimadzu Corporation) Detection: UV 280 nm Temperature: 25°C Sample concentration: 1.5 g / L total amount of lysozyme Sample injection volume: 5 μL

[0087] In Figures 2A and 2B, peak 1 represents tri-PEGylated lysozyme, peak 2 represents di-PEGylated lysozyme, peak 3 represents mono-PEGylated lysozyme, peak 4 represents lysozyme, and peak 5 represents N-hydroxysuccinimide.

[0088] 2A and 2B , the packing materials of Examples 1 to 5 exhibited the peak of tri-PEGylated lysozyme, which exhibited the greatest adsorption due to hydrogen bonding with the residual silanol groups on the porous silica gel surface, and separation of Peak 1 and Peak 2 was confirmed. On the other hand, the packing material packed with the diol-terminated porous silica gel of Comparative Example 1 exhibited insufficient separation of Peak 1 and Peak 2. Furthermore, the packing material of Comparative Example 4, in which n in formula (1) is 61 to 75, also exhibited insufficient separation of Peak 1 and Peak 2. These results demonstrate that a liquid chromatography packing material in which, in formula (1), X is a hydrogen atom, a methyl group, or an ethyl group; Q is a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group; m is 1, 2, or 3; and n is an integer of 1 to 50, exhibits excellent separation properties for polymer compounds.

[0089]

[0090] From the above results, it was found that the packing material for liquid chromatography of this embodiment exhibits excellent separation properties for low molecular weight compounds and hydrophilic polymer compounds at the permeation limit.

Claims

1. A packing material for liquid chromatography comprising a support and a modifier bonded to the support, wherein the modifier has a structure represented by the following formula (1): In formula (1), X represents a hydrogen atom, a methyl group, or an ethyl group; Q represents a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group; m represents 1, 2, or 3; n represents an integer of 1 or more and 50 or less; and the wavy line represents a bonding site between the modifying agent and the carrier.

2. The filler according to claim 1, wherein in formula (1), Q is a urethane bond or an amide bond.

3. The filler according to claim 1 or 2, wherein, in formula (1), X is a hydrogen atom or a methyl group, Q is a urethane bond, and m is 3.

4. A filler according to any one of claims 1 to 3, wherein the carrier is a porous carrier.

5. The filler according to any one of claims 1 to 4, wherein the support is one or more particles selected from the group consisting of silica particles, alumina particles, titania particles, and zirconia particles.

6. A filler according to any one of claims 1 to 5, wherein the carrier has an average particle size (D50) of 1 μm or more and 100 μm or less.

7. A filler according to any one of claims 4 to 6, wherein the average pore diameter of the support is 0.5 nm or more and 200 nm or less.

8. The specific surface area of ​​the carrier is 10 m ２ / g or more 1000m ２ 8. The filler according to claim 4, wherein the filler has a viscosity of 1000 kJ / g or less.

9. A column packed with the packing material for liquid chromatography according to any one of claims 1 to 8.

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