Packers and columns for liquid chromatography

Abstract

The present disclosure aims to provide a filler for liquid chromatography that reduces electrostatic interactions between a polymeric compound, such as a protein, and a silanol of the filler, and has good separation characteristics for low molecular weight compounds with a molecular weight less than or equal to that of the modifier. A filler for liquid chromatography is used, 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 described in the specification).

Description

Technology Field

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

[0002] Size exclusion chromatography (SEC) is a sample separation technique and is used in various fields such as life sciences and medicine. As stationary phases used in SEC, examples include silica gel or surfaces modified with various atomic groups, as described in Non-Patent Literature 1. However, secondary interactions such as electrostatic adsorption by residual silanol on the silica gel surface or hydrophobic adsorption by surface modifiers occur, causing samples such as antibodies, which are in high demand recently, to remain on the stationary phase.

[0003] Patent Document 1 discloses that hydrophobic secondary interactions or electrostatic interactions between a protein and a hydrophobic site of a stationary phase or a silanol are reduced by having hydroxy-terminated polyethylene glycol (PEG) on the surface of the stationary phase. However, there is a problem in that the molecular weight of the modifying agent is large, and the elution sites of substances with lower molecular weights are close together, so low molecular weights cannot be separated, and improvement was required. Prior art literature

[0004] (Patent Document 0001) WO 2022-061039 A

[0005] Andre Striegel, "Modern Size-Exclusion Liquid Chromatography: Practice of Gel Filtration Chromatography" (2nd edition), Publisher: Wiley, 2009. The problem to be solved

[0006] The present disclosure aims to provide a filler for liquid chromatography that reduces electrostatic interactions between a high molecular weight compound, such as a protein, and the silanol of the filler, and has good separation characteristics for low molecular weight compounds with a molecular weight less than or equal to that of the modifier.

[0007] The present disclosure also aims to provide a column packed with the packing material for liquid chromatography. means of solving the problem

[0008] The inventors of the present disclosure, having conducted repeated careful examinations to solve the above problem, discovered the packing material for liquid chromatography described below and completed the present disclosure.

[0009] That is, one aspect of the present disclosure is as shown below.

[0010] [1] A filler for liquid chromatography comprising a carrier and a modifier bonded to the carrier, wherein the modifier has a structure represented by the following formula (1):

[0011]

[0012] In Equation (1),

[0013] X is a hydrogen atom, a methyl group, or an ethyl group, and

[0014] Q is a urethane bond, an amide bond, or a 2-oxopropane-1,3-diyl group, and

[0015] m is 1, 2, or 3, and

[0016] n is an integer between 1 and 50 inclusive, and

[0017] The dashed line indicates the bonding site between the modifier and the carrier.

[0018] [2] The filler described in [1], wherein Q is a urethane bond or an amide bond in formula (1).

[0019] [3] In formula (1), X is a hydrogen atom or a methyl group, and

[0020] The above Q is a urethane bond, and

[0021] The filler described in [1] or [2] above, where m is 3.

[0022] [4] A filler described in any one of [1] to [3], wherein the carrier is a porous carrier.

[0023] [5] A filler described in any one of [1] to [4], wherein the carrier is one or more selected from the group consisting of silica particles, alumina particles, titania particles and zirconia particles.

[0024] [6] A filler described in any one of [1] to [5], wherein the average particle diameter (D50) of the carrier is 1 μm or more and 100 μm or less.

[0025] [7] A filler described in any one of [4] to [6], wherein the average pore diameter of the carrier is 0.5 nm or more and 200 nm or less.

[0026] [8] A filler described in any one of [4] to [7], having a specific surface area of ​​10 m² / g or more and 1000 m² / g or less of the carrier.

[0027] [9] A column filled with a liquid chromatography filler described in any one of [1] to [8]. Effects of the invention

[0028] According to the present disclosure, it is possible to provide a filler for liquid chromatography that reduces electrostatic interactions between a high molecular weight compound, such as a protein, and the silanol of the filler, and has good separation characteristics for low molecular weight compounds with a molecular weight less than or equal to that of the modifier.

[0029] In addition, in the present disclosure, the molecular weight of the polymer compound is not particularly limited and, for example, may be greater 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 also be 100,000,000 or less, 10,000,000 or less, 1,000,000 or less, 100,000 or less, or 1,000 or less. In addition, the molecular weight of the low molecular weight compound is not particularly limited and, for example, may be 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 150 or less, and may also be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more. Brief explanation of the drawing

[0030] FIG. 1a is a diagram showing a chromatogram obtained by separating a low molecular weight compound sample using the filler of an embodiment of the present disclosure. Figure 1b is a diagram showing a chromatogram obtained by separating a low molecular weight compound sample using a filler of a comparative example. FIG. 2a is a diagram showing a chromatogram obtained by separating proteins using the filler of the present disclosure. Figure 2b is a diagram showing a chromatogram obtained by separating proteins using the packing material of a comparative example. Specific details for implementing the invention

[0031] Hereinafter, an example of an embodiment of the filler of the present disclosure is described. In the present disclosure, each component and parameter disclosed in this specification includes any combination, and also includes any combination of upper and lower limits of values ​​disclosed in this specification.

[0032] The present embodiment is a filler for liquid chromatography comprising a carrier and a modifier bonded to the carrier, wherein the modifier has a structure represented by the following formula (1):

[0033]

[0034] 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, n is an integer between 1 and 50, and the dashed line indicates the bonding site between the modifier and the carrier.

[0035] In formula (1), X represents a hydrogen atom, a methyl group, or an ethyl group. For X, a hydrogen atom or a methyl group is preferred because the filler is unlikely to cause hydrophobic interactions and has excellent hydrophilicity.

[0036] 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 direction of the bond may be either way. The amide bond is a bond represented by -NH-CO-, and the direction of the bond may be either way. The 2-oxopropane-1,3-diyl group is a group represented by -CH2-CO-CH2-.

[0037] Among these, regarding Q, it is preferable that it be an amide bond or a urethane bond, and more preferable that it be a urethane bond, as the presence of an amide bond on the surface of the carrier provides an excellent effect in reducing the electrostatic interaction effect of acidic silanol.

[0038] In formula (1), m represents 1, 2, or 3. For the value of m, it is preferable that it be 2 or 3, and more preferable that it be 3, as it has excellent performance in selectively separating low molecular weight compounds and high molecular weight compounds through hydrophobic interactions.

[0039] In Equation (1), n ​​represents an integer from 1 to 50. 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 high molecular weight compound, it is preferable that the value be an integer from 8 to 22, and more preferable that the value be an integer from 8 to 10, as this provides an excellent shielding effect in blocking access to surface silanol. When the compound is a low molecular weight compound, it is preferable that the value be an integer from 40 to 50.

[0040] In addition, regarding the modifier represented by formula (1), it is preferable that it be a structure represented by formula (2) or (3) in that it has excellent hydrophilicity and stereoexclusion effects due to the hydration of polyethylene glycol (PEG).

[0041]

[0042]

[0043] Here, the dashed line indicates the bonding site between the modifier and the carrier.

[0044] When X in formula (1) is a hydrogen atom, the hydroxyl group-terminated PEG is difunctional, so the carrier of this embodiment may include a cross-linked PEG-containing group represented by formula (4).

[0045]

[0046] Here, the dashed line indicates the bonding site between the modifier and the carrier.

[0047] It is preferable that the average particle diameter of the carrier of this embodiment (hereinafter also referred to as 'D50') be 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, and may also 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 limits may be any combination. Accordingly, for example, the average particle diameter 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 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 less, It may be 1.7㎛ or more and 2.0㎛ or less, 1.7㎛ or more and 3.0㎛ or less, 1.7㎛ or more and 4.0㎛ or less, 1.7㎛ or more and 5.0㎛ or less, 1.7㎛ or more and 10㎛ or less, or 1.7㎛ or more and 50㎛ or less.

[0048] Since the particle diameter of the filler according to the present embodiment corresponds to the particle diameter of the carrier used, a carrier with a desired average particle diameter can be selected. Generally, in the manner in which the modifier is bonded to the carrier, there is no change in the average particle diameter beyond the error before and after bonding, so the average particle diameter of the filler can be considered to be the same as the average particle diameter of the carrier used.

[0049] Accordingly, it is preferable that the average particle diameter of the filler according to the present embodiment be 1 μm or more and 100 μm or less. For example, the average particle diameter of the filler 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, and may also 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 limits can be any combination. Accordingly, for example, the average particle diameter 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 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, and 1.7 µm or more and 1.9 µm It may be 1.7㎛ or more and 2.0㎛ or less, 1.7㎛ or more and 3.0㎛ or less, 1.7㎛ or more and 4.0㎛ or less, 1.7㎛ or more and 5.0㎛ or less, 1.7㎛ or more and 10㎛ or less, or 1.7㎛ or more and 50㎛ or less.

[0050] The average particle diameter of this embodiment can be obtained by the Coulter method using a general particle size distribution measuring device (e.g., product name: Multisizer3, manufactured by BECKMAN COULTER). In addition, the Coulter method is generally recognized as an electrical resistance method. The measurement conditions may be exemplified as follows.

[0051] <Measurement Sample>

[0052] Silica gel (measurement sample): approx. 0.0010g

[0053] <Preprocessing>

[0054] Electrolyte: ISOTON-II (Beckman Coulter product)

[0055] Ultrasonic Disperser: BRANSONIC M2800-J

[0056] Frequency: 40kHz

[0057] Processing time: 1 minute

[0058] Average Particle Diameter Measurement

[0059] For silica gel (measurement sample) after pretreatment, measurements are performed using a particle size distribution measuring device (Multisizer3, manufactured by BECKMAN COULTER) under conditions where the aperture diameter is 50 μm and the number of measured particles is 50,000 counts, and the volume average particle diameter can be obtained from the volume particle size distribution of the sample particles.

[0060] The carrier of the present embodiment is not particularly limited, but examples include inorganic particles, resin particles, etc., and more specifically, silica particles, alumina particles, titania particles, zirconia particles, magnesia particles, hydroxyapatite particles, silicon particles, polyacrylic acid particles, polymethacrylic acid particles, nylon particles, or polyamide particles.

[0061] Regarding the above carrier, it is preferable that it be one or more selected from the group consisting of silica particles, alumina particles, titania particles, and zirconia particles in terms of excellent mechanical strength, and it is more preferable that it be silica particles.

[0062] As a carrier of the present embodiment, it may be porous or non-porous, but it is preferable to be a porous particle in that it provides excellent separation based on molecular weight distribution as a filler for size exclusion chromatography. That is, regarding the particles, it is more preferable that one or more are selected from the group consisting of porous silica particles, porous alumina particles, porous titania particles, and porous zirconia particles, and it is more preferable that they be porous silica particles.

[0063] In addition, examples of porous particles include silica monoliths, alumina monoliths, silica-alumina monoliths, polymethylsilsesquioxane monoliths, titanium oxide monoliths, polyacrylamide monoliths, or polymethacrylic acid ester monoliths. Monoliths are generally recognized as porous bodies.

[0064] Preferably, the average pore diameter of the carrier in this embodiment is 0.5 nm or more and 200 nm or less. For example, the average pore diameter of the carrier 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, and may also 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. These upper and lower limits may be any combination. Therefore, for example, the average pore diameter of the carrier is 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, 19 nm or more It may be 30 nm or less.

[0065] In addition, it is preferable that the specific surface area of ​​the carrier in this embodiment be 10 m² / g or more and 1000 m² / g or less. For example, the specific surface area of ​​the carrier may be 10 m² / g or more, 20 m² / g or more, 30 m² / g or more, 40 m² / g or more, 50 m² / g or more, or 100 m² / g or more, 150 m² / g or more, or 200 m² / g or more, and may also be 1000 m² / g or less, 900 m² / g or less, 800 m² / g or less, 700 m² / g or less, 600 m² / g or less, 500 m² / g or less, 400 m² / g or less, 300 m² / g or less, or 250 m² / g or less. These upper and lower limits may be any combination. Accordingly, for example, the specific surface area of ​​the carrier is 10㎡ / g or more and 250㎡ / g or less, 10㎡ / g or more and 300㎡ / g or less, 10㎡ / g or more and 400㎡ / g or less, 10㎡ / g or more and 500㎡ / g or less, 10㎡ / g or more and 600㎡ / g or less, 50㎡ / g or more and 250㎡ / g or less, 50㎡ / g or more and 300㎡ / g or less, 50㎡ / g or more and 400㎡ / g or less, 50㎡ / g or more and 500㎡ / g or less, 50㎡ / g or more and 600㎡ / g or less, 100㎡ / g or more and 250㎡ / g or less, 100㎡ / g or more and 300㎡ / g or less, 100㎡ / g or more and 400㎡ / g or less, 100㎡ / g or more and 500㎡ / g or less, It may be 100㎡ / g or more and 600㎡ / g or less, 200㎡ / g or more and 250㎡ / g or less, or 200㎡ / g or more and 300㎡ / g or less.

[0066] Since the specific surface area of ​​the filler according to the present embodiment corresponds to the specific surface area of ​​the carrier used, a carrier with a desired specific surface area can be selected. Generally, in the manner in which a modifier is bonded to a carrier, there is no change in the specific surface area beyond the margin of error before and after bonding, so the specific surface area of ​​the filler can be considered to be the same as the specific surface area of ​​the carrier used.

[0067] Accordingly, it is preferable that the specific surface area of ​​the filler in this embodiment be 10 m² / g or more and 1000 m² / g or less. For example, the specific surface area of ​​the filler may be 10 m² / g or more, 20 m² / g or more, 30 m² / g or more, 40 m² / g or more, 50 m² / g or more, 100 m² / g or more, 150 m² / g or more, or 200 m² / g or more, and may also be 1000 m² / g or less, 900 m² / g or less, 800 m² / g or less, 700 m² / g or less, 600 m² / g or less, 500 m² / g or less, 400 m² / g or less, 300 m² / g or less, or 250 m² / g or less. These upper and lower limits may be any combination. Accordingly, for example, the specific surface area of ​​the filler is 10㎡ / g or more and 250㎡ / g or less, 10㎡ / g or more and 300㎡ / g or less, 10㎡ / g or more and 400㎡ / g or less, 10㎡ / g or more and 500㎡ / g or less, 10㎡ / g or more and 600㎡ / g or less, 50㎡ / g or more and 250㎡ / g or less, 50㎡ / g or more and 300㎡ / g or less, 50㎡ / g or more and 400㎡ / g or less, 50㎡ / g or more and 500㎡ / g or less, 50㎡ / g or more and 600㎡ / g or less, 100㎡ / g or more and 250㎡ / g or less, 100㎡ / g or more and 300㎡ / g or less, 100㎡ / g or more and 400㎡ / g or less, 100㎡ / g or more and 500㎡ / g or less, It may be 100㎡ / g or more and 600㎡ / g or less, 200㎡ / g or more and 250㎡ / g or less, or 200㎡ / g or more and 300㎡ / g or less.

[0068] The average pore diameter and specific surface area of ​​the carrier of this embodiment can be measured by the gas adsorption method using a general gas adsorption amount measuring device (product name: BELSORP MINI II, MicrotracBEL product).

[0069] The gas adsorption method is a method of adsorbing gas by changing the pressure on a porous sample, measuring the amount of adsorption at that time, and calculating the specific surface area, pore volume, pore distribution, etc. from an adsorption-desorption isotherm plotting the relative pressure (the ratio of the pressure at adsorption equilibrium state to the saturated vapor pressure) and the amount of adsorption, and JIS Z8831-2 (mesopores and macropores) or JIS Z8831-3 (micropores) specified for the pore diameter distribution and pore characteristics of a powder (solid) may be applied.

[0070] The filler of the present embodiment can be prepared by reacting the aforementioned carrier with a compound represented by formula (5).

[0071]

[0072] In Equation (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 between 1 and 50. Also, R1 is OCH3, OC2H5, OC3H7, OCH(CH3)2, OC2H4OCH3, OC(=O)CH3, N(CH3)2, or Cl, and R 2 and R 3 Each is independently selected from the group consisting of H, CH3, C2H5, C3H7OCH3, OC2H5, OC3H7, OCH(CH3)2, OC2H4OCH3, OC(=O)CH3, N(CH3)2 and Cl.

[0073] As a solvent for binding a modifying agent to a carrier, it is not particularly limited, but may be one or more selected from the group consisting of, for example, 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, acetanitrile, dimethyl sulfoxide, N,N-dimethylformamide, water, and an aqueous solution containing a salt of 0.001 to 5.0 mol / ℓ.

[0074] In the above aqueous solution containing a salt of 0.001 to 5.0 mol / ℓ, 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.

[0075] The temperature at which the modifying agent is bound to the carrier is not particularly limited, but may be 0°C or higher and 300°C or lower, and preferably is the reflux temperature of the solvent.

[0076] As for the concentration of the modifier, it is preferable to be 0.01 mmol or more and 4.0 mmol or less per 1 g of carrier. For example, the modifier 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, and may also be 4.0 mmol or less, 3.0 mmol or less, 2.5 mmol or less, or 2.0 mmol or less. These upper and lower limits may be any combination. Accordingly, for example, the concentration of the modifier 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, and 1.0 mmol or more and 2.0 mmol or less, per 1 g of carrier.

[0077] As a catalyst for bonding a modifying agent to a carrier, it is not particularly limited, but may be 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.

[0078] Also, regarding the compound represented by formula (5), a commercially available product may be used, or it may be manufactured according to a generally known method.

[0079] The packing material of the present embodiment can be used for chromatography after being packed, for example, into a column, and it is preferable to use it for size exclusion chromatography. A retention factor (k) can be used as an indicator to evaluate the separation performance of a low molecular weight compound sample in the packing material of the present embodiment. The retention factor (k) is defined as follows.

[0080] (Maintenance factor)

[0081] Retention coefficient k = (t R -t0) / t0

[0082] t R : Sample elution time

[0083] t0: Elution time of non-ferrous sample

[0084] In this embodiment, t0 is the dissolution time of uracil.

[0085] In this embodiment, based on the large value of the retention factor, the separation performance of low molecular weight compound samples in the packing agent of this embodiment is evaluated as improved. Also, the elution time is defined as the time when the sample is injected as 0, and the time when the perpendicular line is drawn from the peak top of each sample in the chromatogram as the elution time.

[0086] As an indicator for evaluating the effect of reducing electrostatic interaction between the polymer compound and the silanol of the filler in the filler of the present embodiment, the peak area and the ratio of the peak to the total area may be used. The ratio of the peak to the total area is defined as follows.

[0087] (Ratio of peaks to total area)

[0088] Ratio of peak to total area = A1 / A all

[0089] A1: Peak area of ​​the sample

[0090] A all : Peak area of ​​the entire peak

[0091] In this embodiment, A1 is defined as the peak area of ​​the triPEGylated lysozyme that is most adsorbed by hydrogen bonding with the residual silanol group on the surface of the porous silica gel.

[0092] In this embodiment, based on the large peak area and the large ratio of the peak to the total area, it is evaluated that the effect of reducing electrostatic interaction between the polymer compound and the silanol of the filler in this embodiment is enhanced.

[0093] The samples to be separated by the filler of the present embodiment are not particularly limited, but examples include sugars, proteins, PEGylated proteins, peptides, oligonucleotides, nucleic acids, antibodies, antibody-drug conjugates, enzymes, extracellular vesicles, adeno-associated viruses, lipid nanoparticles, ribosomes, or lentiviruses.

[0094] [Example]

[0095] The present disclosure will be explained below by way of examples. However, the present disclosure is not limited to these.

[0096] (Particle diameter distribution measurement)

[0097] The volumetric particle diameter 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), BECKMAN COULTER product), and the median diameter (D50) was measured from this. Prior to the measurement, the sample was suspended in pure water and dispersed using an ultrasonic homogenizer for 1 minute as a pretreatment.

[0098] (Specific surface area measurement)

[0099] Using a gas adsorption measurement device (Product name: BELSORP MINI II, MicrotracBEL product), the adsorption-desorption isotherm due to nitrogen adsorption was measured using the fixed-dose method, and the specific surface area a was determined by the BET multi-point method. s , BET was calculated.

[0100] (Measurement of pore diameter)

[0101] Using a gas adsorption measurement device (Product name: BELSORP MINI II, MicrotracBEL product), the adsorption-desorption isotherm due to nitrogen adsorption was measured using the constant volume method, and the total pore volume V was determined from the pore distribution on the desorption side of the BJH method. p was calculated. The average pore diameter D is 4Vp / a s, BET It was calculated by.

[0102] Example 1

[0103] (1-1) Synthesis of Modifying Agents

[0104] 2.47 g of (3-isocyanatopropyl)trimethoxysilane (CAS RN 15396-00-6, product of Tokyo Chemical Industry Co., Ltd.) and 5.29 g of polyethylene glycol monomethyl ether 400 (CAS RN 9004-74-4, product of Tokyo Chemical Industry Co., Ltd., average molecular weight 380–420) were mixed in a glass vial. The glass vial was heated to 75°C in an oil bath and maintained for 12 hours while stirring to obtain a modifying agent.

[0105] (1-2) Binding to the carrier

[0106] 4.00 g of porous silica gel (D50 1.78 μm, average pore diameter 20.1 nm, specific surface area 231 m² / g) vacuum-dried overnight at 110°C and 80 ml of a 10 mMol / ℓ aqueous sodium phosphate solution (pH 6.0) were mixed, and the porous silica gel was dispersed by irradiating it with ultrasound for 1 minute.

[0107] Next, the modifying agent obtained in (1-1) was added at a concentration 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 while stirring. After the silane coupling reaction was completed, the obtained powder was filtered and recovered, and the filler of the present example was obtained by washing it 5 times with 100 ml of hot water at 70°C.

[0108] It can be presumed that the filler in question has the following structure.

[0109]

[0110] Example 2

[0111] In (1-2), the filler of this example was obtained by adding the modified agent obtained in (1-1) in an amount of 2 mmol per 1 g of porous silica gel, in the same manner as in Example 1.

[0112] It can be presumed that the filler in question has the same structure as Example 1. Also, in the case of Example 2, since the amount of modifier added is greater than in the case of Example 1, it can be presumed that the density of modifiers that can be generated (i.e., modifiers bound to the carrier) is higher than in the case of Example 1.

[0113] Example 3

[0114] The filler of this example was obtained by performing the same procedure as in Example 2, except that 4.40 g of PEG400 (CAS RN 25322-68-3, FUJIFILM Wako Pure Chemical Corporation, average molecular weight 360-440) was used instead of 2.06 g of (3-isocyanatopropyl)trimethoxysilane (product of Tokyo Chemical Industry Co., Ltd.) and polyethylene glycol monomethyl ether 400.

[0115] It can be presumed that the filler in question has the following structure. Additionally, since the modifier contains a modifier having a cross-linked structure when the modifier is synthesized, it can be presumed that a structure as follows can be generated.

[0116]

[0117] Example 4

[0118] The filler of the present example was obtained by performing the same procedure as in Example 3, except that 40 ml of a 10 ml / ℓ ammonium acetate aqueous solution (pH 9.5) was mixed instead of a 10 ml / ℓ sodium phosphate aqueous solution (pH 6.0).

[0119] It can be presumed that the filler in question has a structure similar to that of Example 3.

[0120] Example 5

[0121] The filler of this example was obtained by performing the same procedure as in Example 3, except that 2.90 g of (3-isocyanatopropyl)trimethoxysilane (product of Tokyo Chemical Industry Co., Ltd.) and 42.03 g of PEG2000 (CAS RN 25322-68-3, product of FUJIFILM Wako Pure Chemical Corporation, average molecular weight 1,800–2,200) were used instead of PEG400. It can be presumed that the filler has the following structure. Furthermore, this structure can be presumed as in the case of Example 3.

[0122]

[0123] Comparative Example 1

[0124] A diol-terminated porous silica gel-packed chromatography column (product name: TSKgel UP-SW3000-LS, inner diameter 4.6 mm, length 15 cm, manufactured by TOSOH CORPORATION) was used.

[0125] Comparative Example 2

[0126] 5.00 g of porous silica gel (D50 1.78 μm, average pore diameter 20.1 nm, specific surface area 231 m² / g) vacuum-dried overnight at 110°C and 100 ml of a 10 mMol / ℓ aqueous sodium phosphate solution (pH 6.0) were mixed, and the porous silica gel was dispersed by irradiating it with ultrasound for 1 minute.

[0127] Next, 3-[methoxy(polyethyleneoxy)6-9propyl]trimethoxysilane (CAS RN 65994-07-2, Gelest product, tech-90) was added at a concentration 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 while stirring. After the silane coupling reaction was completed, the obtained powder was filtered and recovered, and the filler of the present comparative example was obtained by washing it five times with 100 ml of hot water at 70°C.

[0128] It can be presumed that the filler in question has the following structure.

[0129]

[0130] Comparative Example 3

[0131] Porous silica gel without added modifiers (D50 1.78㎛, average pore diameter 20.1 nm, specific surface area 231 m² / g) was used as the filler for the comparative example.

[0132] Comparative Example 4

[0133] The filler of the present Comparative Example was obtained by performing the same procedure as in Example 3, except that 2.89 g of (3-isocyanatopropyl)trimethoxysilane (product of Tokyo Chemical Industry Co., Ltd.) and 63.02 g of PEG4000 (CAS RN is 25322-68-3, product of FUJIFILM Wako Pure Chemical Corporation, average molecular weight 2,700–3,300) were used instead of PEG400.

[0134] Comparative Example 5

[0135] The filler of the present comparative example was obtained by performing the same procedure as in comparative example 4, except that a porous silica gel with a D50 of 3.82 μm, an average pore diameter of 111 nm, and a specific surface area of ​​28 m² / g was used.

[0136] The packing materials obtained in Examples 1 to 5 and Comparative Examples 2 to 5 were packed into a liquid chromatography column with an inner diameter of 4.6 mm and a length of 15 cm. These packing materials are shown in Table 1.

[0137]

[0138] [Separation of low molecular weight compound samples]

[0139] For 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.

[0140] Let the elution time of uracil be t0, and the elution time of each sample be t R If so, the retention coefficient k is (t R It is expressed as -t0) / t0. The obtained chromatograms are shown in Figures 1a and 1b, and the retention factor k of each substance is shown in Table 2.

[0141] (Measurement conditions)

[0142] Eluent: 100 mM / L phosphate buffer (pH 5.0), 100 mM / L sodium sulfate and 0.05% sodium azide

[0143] Flow rate: 0.35 ml / min

[0144] Detector: SPD-20A (Shimadzu Corporation)

[0145] Detection: UV 280 nm

[0146] Temperature: 25℃

[0147] 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

[0148] Sample injection volume: 5 µl

[0149] 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.

[0150] From Table 2, it was confirmed that the retention factor k of Examples 1 to 5 was greater than that of Comparative Examples 1, 2, 3, and 5 for any low molecular weight compound sample, and that the filler of the present embodiment is excellent for separating low molecular weight compound samples located near the penetration limit. On the other hand, in Comparative Example 2, which does not contain urethane bonds in the modifier, it was confirmed that peak 3 of p-aminobenzoic acid and peak 4 of p-hydroxybenzoic acid overlapped, making clear separation impossible. In addition, in Comparative Example 3, which is a 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, it was confirmed that the retention factor k was smaller compared to Examples 1 to 5, and the separation state was not good. In addition, 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, and separation deteriorated. From this, it was found that a packing material for liquid chromatography in which X is a hydrogen atom, a methyl group, or an ethyl group in Formula (1), 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 between 1 and 50, exhibits excellent separation properties for low molecular weight compounds. Furthermore, since the separation properties for low molecular weight compounds are improved because Q in Formula (1) is a urethane bond to the oxygen atom, it can be understood that an amide bond is required on the surface of the carrier.

[0151]

[0152] [Isolation of Proteins]

[0153] Separation of PEGylated proteins that cause adsorption by hydrogen bonding with residual silanol groups on the surface of porous silica gel was attempted using columns of Examples 1 to 5, which showed good separation ability in low molecular weight compound samples, and Comparative Examples 1 and 4, which had no peak overlap.

[0154] Lysozyme (product of FUJIFILM Wako Pure Chemical Corporation, derived from egg white, molecular weight: 14,307) was used as an example of a protein, 15 mg of the said lysozyme, 16 mg of SUNBRIGHT ME-050CS (product of NOF CORPORATION), and 10 mL of 10 mM / L phosphate buffer (pH 6.5) were mixed and stirred at 25°C for 2 hours to obtain PEGylated lysozyme.

[0155] For the liquid chromatography columns obtained in Examples 1 to 5 and Comparative Examples 1 and 4, the PEGylated lysozyme was separated under the following measurement conditions. The obtained chromatograms are shown in FIGS. 2a and 2b. In addition, Table 3 shows the peak area of ​​peak 1, which is triPEGylated lysozyme, and the ratio of peak 1 to the total area.

[0156] (Measurement conditions)

[0157] Eluent: 100 mM / L phosphate buffer (pH 6.7), 100 mM / L sodium sulfate and 0.05% sodium azide

[0158] Flow rate: 0.35 ml / min

[0159] Detector: SPD-20A (Shimadzu Corporation)

[0160] Detection: UV 280 nm

[0161] Temperature: 25℃

[0162] Sample concentration: 1.5 g / ℓ Total amount of lysozyme

[0163] Sample injection volume: 5 µl

[0164] In Figures 2a and 2b, peak 1 represents triPEGylated lysozyme, peak 2 represents diPEGylated lysozyme, peak 3 represents monoPEGylated lysozyme, peak 4 represents lysozyme, and peak 5 represents N-hydroxysuccinimide.

[0165] From FIGS. 2a and 2b, in the fillers of Examples 1 to 5, the peak of triPEGylated lysozyme, which is adsorbed by hydrogen bonding with residual silanol groups on the surface of the porous silica gel, was identified, and the separation of peak 1 and peak 2 was confirmed. On the other hand, in the filler packed with the diol-terminated porous silica gel of Comparative Example 1, the separation of peak 1 and peak 2 was insufficient. Also, in the filler of Comparative Example 4, where n in Formula (1) is 61 or more and 75 or less, the separation of peak 1 and peak 2 was insufficient. 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 between 1 and 50, exhibits excellent separation properties for polymer compounds.

[0166]

[0167] From the above results, it was found that the packing agent for liquid chromatography according to the present embodiment exhibits excellent separation properties for low molecular weight compounds and hydrophilic polymer compounds located at the penetration limit.

Claims

Claim 1 A filler for liquid chromatography comprising a carrier and a modifying agent bound to the carrier, wherein the modifying agent has a structure represented by the following formula (1): 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, n is an integer between 1 and 50, and the dashed line indicates the bonding site between the modifier and the carrier. Claim 2 A filler for liquid chromatography according to claim 1, wherein in formula (1), Q is a urethane bond or an amide bond. Claim 3 A filler for liquid chromatography 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. Claim 4 A filler for liquid chromatography according to any one of claims 1 to 3, wherein the carrier is a porous carrier. Claim 5 A filler for liquid chromatography according to any one of claims 1 to 4, wherein the carrier is one or more selected from the group consisting of silica particles, alumina particles, titania particles and zirconia particles. Claim 6 A filler for liquid chromatography according to any one of claims 1 to 5, wherein the average particle diameter (D50) of the carrier is 1 μm or more and 100 μm or less. Claim 7 A filler for liquid chromatography according to any one of claims 4 to 6, wherein the average pore diameter of the carrier is 0.5 nm or more and 200 nm or less. Claim 8 A filler for liquid chromatography according to any one of claims 4 to 7, wherein the specific surface area of ​​the carrier is 10 m² / g or more and 1000 m² / g or less. Claim 9 A column packed with a liquid chromatography packing material as described in any one of claims 1 to 8.

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