JP2001318086A - Filler for liquid chromatography used for separating protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a filler for liquid chromatography used for separating protein in a short time. SOLUTION: This filler for liquid chromatography used for separating protein is characterized by comprising a porous high polymer obtained by water- suspension polymerizing two or more kinds of ethylenic unsaturated monomers selected from among the three kinds A, B, and C mentioned below in the existence of a solvent that is solvent of monomers but not solvent of their polymers. A: CH2=CR1R2, where R1 designates hydrogen or a methyl group and R2 designates either a 2-hydroxy ethyl group or an amide group. B: (Shown in the formula), where, R3 and R4 designate hydrogen or a methyl group, and n designates an integer from 1 to 20. C: CH2=CHR5R6, where R5 designates hydrogen or a methyl group, and R6 designates a phenyl group, a glycigyl group, and acryl ester group, a nitrile group, or a monovinyl-substitutional phenyl group. As for the monomers classified into the groups A, B, and C, the selection of two or more kinds may be made from the same group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a packing material for liquid chromatography (HPLC) used for separating proteins.

[0002]

2. Description of the Related Art In a method for measuring a drug in a biological sample, HP is used.
The LC method has the advantages of higher accuracy and easier separation from metabolites than other measurement methods. However, since proteins present in biological samples are irreversibly adsorbed or deposited on the analytical column and inhibit the separation of drugs,
It was difficult to analyze a drug by directly injecting a biological sample into an analytical column of an HPLC device. Therefore, conventionally, when analyzing a drug in a biological sample, a pretreatment operation such as an operation of removing a protein or an extraction operation with an organic solvent has been required. However, these pre-processing operations are very time consuming and often cause measurement errors.
Furthermore, the organic solvents used are harmful substances,
There are drawbacks such as a large cost involved in their disposal. Therefore, there has been an extremely strong demand for a packing material for HPLC that efficiently separates proteins and the like.

[0003]

As a filler for HPLC for separating high-molecular substances such as proteins, it has been used as a filler for HPLC.
Silica-based fillers have been developed, but the separation efficiency is poor and not always satisfactory. Therefore, an object of the present invention is to provide a packing material for HPLC that efficiently separates proteins.

[0004]

SUMMARY OF THE INVENTION In view of the above-mentioned drawbacks of the prior art, the present invention has been made as a result of intensive studies for the purpose of providing a packing material for HPLC having better separation performance for proteins. It is completed.

In the present invention, two or more types of ethylenically unsaturated monomers selected from the following three types of ethylenically unsaturated monomers consisting of the following A, B and C are dissolved in the monomer. An object of the present invention is to provide a packing material for HPLC used for protein separation characterized by comprising a porous polymer obtained by subjecting a polymer to aqueous suspension polymerization in the presence of an insoluble organic solvent. A CH ₂ = CR ₁ R ₂ wherein R ₁ represents hydrogen or a methyl group, and R ₂ represents either a 2-hydroxyethyl group or an amide group. Here, R ₃ and R ₄ represent hydrogen or a methyl group, and n represents an integer of 1 to 20. C CH ₂ ＣＨCHR ₅ R ₆ wherein R ₅ represents hydrogen or a methyl group, and R ₆ represents any one of a phenyl group, a glycidyl group, an acrylester group, a nitrile group, and a monovinyl-substituted phenyl group. The monomers classified into the groups A, B, and C do not prevent two or more kinds from being selected from the same group.

[0006] In the present invention, HPLC means that a sample and an eluent are pressure-fed by a constant flow pump into a column filled with a packing material, and the sample is adsorbed and desorbed in the column, and the sample is subjected to its characteristics. Means an analysis method in which each component is separated according to and passed through the column, and the separation state is detected by an ultraviolet detector, a differential refraction detector, or the like.In the present invention, a protein is applied as a sample, and elution is performed. Water or an aqueous solution is used as the liquid. The filler used in the present invention has a function that water or an aqueous solution can be used as an eluent, that a protein is eluted without being retained, and that various drugs are retained. By using an HPLC packing material having such a function and changing the ionic strength of the eluent according to, for example, a change in the salt concentration, HPLC is used to effectively separate proteins which were conventionally considered difficult to separate. It can be done. More specifically, to create an excellent HPLC packing material that is effective in removing contaminating proteins that is performed as a pretreatment for the measurement of drugs in human serum / plasma, various experimental animal sera / plasma, meat and fish meat. Is also an important object of the present invention.

[0007] Among the above-mentioned functions of the filler used in the present invention, with respect to the fact that water or an aqueous solution can be used as an eluent and that the protein is eluted without being retained in the filler, the following is essential. It is. Having an affinity for water in the structure of the synthetic polymer polymer constituting the filler,
It also has a hydrophilic group having a steric hindrance to proteins and a molecular size exclusion function, and it is first necessary that the surface of the filler be wet with water and that the protein be eluted without being retained by the filler. Further, it is necessary that a non-dissociative hydrophilic group such as an alcoholic hydroxyl group and an ethylene oxide group among the hydrophilic groups be present on the surface. In addition, dissociative hydrophilic groups such as carboxyl groups and amino groups have the effect of imparting hydrophilicity.However, if these groups are present, the ion exchange function acts concurrently with the chromatography function, and the separation behavior is complicated. Undesirably results.
The separation effect of the present invention is believed to be based solely on hydrophobic interactions. The hydrophobic interaction is a statistical thermodynamic interaction whose main effect is an increase in entropy, unlike an interaction mainly based on binding energy, such as electrostatic bonding or hydrogen bonding. In order to cause such an interaction, it is necessary that a hydrophobic group such as an alkyl group, an alkylene group, or a phenyl group be present in the synthetic polymer constituting the filler.
Therefore, in the synthetic polymer constituting the filler used in the present invention, it is essential that both the non-dissociable hydrophilic group and the hydrophobic group described above are present in the molecular structure thereof, Furthermore, it is necessary that both of them exist with good lance.

In preparing the filler used in the present invention, a plurality of types of polymerizable monomers are polymerized to contain both the above-mentioned hydrophilic group and hydrophobic group in a well-balanced manner.
The object can be achieved by obtaining a synthetic high molecular polymer having the above-mentioned characteristics. Specifically, a method of copolymerizing a highly hydrophilic monomer and a hydrophobic monomer or a relatively strong hydrophobic monomer by an aqueous suspension polymerization method is often adopted. Examples of the highly hydrophilic monomer include tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, and acrylamide. Examples of the hydrophobic monomer include styrene, divinylbenzene, vinyl acetate, glycidyl acrylate, and glycidyl methacrylate. Examples of the relatively strong hydrophobic monomer include ethylene glycol diacrylate, ethylene glycol dimethacrylate, diethylene glycol diaacrylate, diethylene glycol dimethacrylate, tetramethylol methane triacrylate, and tetramethylol methane trimethacrylate. In the ethylenically unsaturated monomer belonging to the group A according to claim 1 of the claims, the smaller the value of the integer n in the chemical structural formula, the stronger the hydrophobicity and the larger the value of the integer n. The hydrophilicity tends to increase. Therefore, it is desirable to estimate the value of the integer n and select an appropriate monomer while taking into account the performance to be satisfied by the filler to be obtained.

In the present invention, from the viewpoint of improving the balance between both the hydrophilic group and the hydrophobic group present in the synthetic high molecular polymer, one or more monomers having a strong hydrophilic property are used. The use of a synthetic polymer obtained by copolymerizing at least one kind of relatively strong monomer as a filler for HPLC is most preferable because it also complies with the gist of the present invention. In the present invention, a monomer having strong hydrophilicity is about 5% to 30% by weight of all monomers, and a monomer having relatively strong hydrophobicity is 30% to 70% by weight.
By setting the monomer composition distribution occupied by the monomer having a strong hydrophobicity to the extent, a filler for HPLC with good performance can be obtained. For polymerization or copolymerization as a method for obtaining the HPLC filler of the present invention, it is most practical to use a conventionally known aqueous suspension polymerization method. At that time, an organic solvent in which the monomer is dissolved but the polymer is not dissolved is added, and after the polymerization is completed, the above-mentioned organic solvent is removed from the particles by heating or washing, etc., and the resulting spherical polymer becomes porous, For this reason, the surface area of the filler increases, so that the separation efficiency can be effectively improved. As the organic solvent, in principle, all organic solvents that dissolve the monomer but do not dissolve the polymer can be used, for example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene, dodecylbenzene, Hexane, heptane,
Examples include saturated hydrocarbons such as octane and decane, and alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol. Although the addition amount is not limited at all, based on many years of empirical knowledge in the art, 15 to 100 parts by weight of the monomer
Parts by weight to 200 parts by weight, more preferably 3 parts by weight.
0 to 150 parts by weight. Further, as a requirement for being a filler for HPLC, it is required that the filler has excellent mechanical strength and low swelling property. Therefore, it is desirable that the filler be spherical and chemically crosslinked. Therefore, it is desirable that the monomer to be subjected to polymerization or copolymerization is a polyfunctional one or contains a polyfunctional one.

As a method of aqueous suspension polymerization, for example, a solution obtained by uniformly dissolving the above-mentioned monomer, organic solvent and radical polymerization initiator is added to an aqueous phase, and the temperature is raised to a constant temperature while stirring. Then, the temperature is maintained at this temperature for a certain period of time. At this time, the polymerization temperature is 30 ° C to 100 ° C, more preferably 45 ° C to 85 ° C, and the polymerization time is 2 hours.
Hours to 100 hours, more preferably 4 hours to 5 hours
0 hours. At this time, a suspension stabilizer may be added to the polymerization system in order to improve the dispersibility of the raw material composition for polymerization. The suspension stabilizer is not particularly limited and includes, for example, water-soluble polymers such as polyvinyl alcohol, starch, methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, and sodium polymethacrylate, barium sulfate, calcium sulfate, aluminum sulfate, and calcium carbonate. , Talc, clay, diatomaceous earth, metal oxide powder and the like. The amount of the suspension stabilizer is usually from 0.01% by weight to 50% by weight, more preferably from 0.05% by weight to 35% by weight, based on the total amount of the polymerizable monomer. Examples of the radical polymerization initiator include oil-soluble organic peroxides and azonitrile compounds used in ordinary suspension polymerization. Examples of the organic peroxide include acetyl peroxide, decanoyl peroxide, lauroyl peroxide, benzoyl peroxide, o-methoxy benzoyl peroxide, p-chlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, and carbonic acid peroxide. Diisopropyl, di-2-ethylhexyl peroxide, acetylcyclohexylsulfonyl peroxide, t-butyl perisobutyrate, t-butyl perivalate, t-butyl per-2-ethylhexanoate, t-butyl peroxide, t-butyl peroxide t-butyl cumyl, dicumyl peroxide, methyl ethyl ketone peroxide, cumene hydroperoxide, t-butyl hydroperoxide and the like. Examples of the azonitrile compounds include 2,2'-azobisisobutyronitrile and 2,2 '
-Azobis-2,4'-dimethylvaleronitrile, 2,
2'-azobismethylbutyronitrile, 2,2'-azobis-2,3-dimethylbutyronitrile, 2,2'-azobis-2,3,3-trimethylbutyronitrile, 2,
2'-azobis-2-isopropylbutyronitrile,
4,4-azobis-4-cyanovaleric acid, dimethyl-
Examples thereof include 2,2-azobisisobutyrate and 2,2′-azobismethylheptonitrile. If such a radical polymerization initiator is too small, the polymerization rate may decrease, and even if it is too large, an effect of a certain level or more cannot be obtained.
Usually, it is used in the range of 0.05% by weight to 30% by weight based on the total amount of the monomers, and more preferably 0.2% by weight.
% By weight to 10% by weight.

The synthetic polymer particles obtained by suspension polymerization based on the gist of the present invention are purified by removing impurities and other components other than the particles, and then dried by heating or the like. Then, it is determined whether or not the dry particles are wetted by dispersing the particles in water. Particles that do not get wet with water are excluded from the scope of the present invention because they cannot be used for HPLC using water or an aqueous solution as an eluent. Next, the synthetic polymer particles having a particle diameter of several micrometers to several hundreds of micrometers obtained by removing fine particles and coarse particles are packed into a stainless steel column by pumping the ion exchange water with a constant flow pump using a constant flow pump. Provide a practical test.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention basically has the following configuration. That is, first, two or more types of ethylenically unsaturated monomers selected from the following three types of ethylenically unsaturated monomers consisting of A, B, and C are dissolved in the monomer. A filler for liquid chromatography, which is used for separating a protein consisting of a porous polymer obtained by aqueous suspension polymerization in the presence of an organic solvent in which the polymer does not dissolve. A CH ₂ = CR ₁ R ₂ wherein R ₁ represents hydrogen or a methyl group, and R ₂ represents either a 2-hydroxyethyl group or an amide group. Here, R ₃ and R ₄ represent hydrogen or a methyl group, and n represents an integer of 1 to 20. C CH ₂ ＣＨCHR ₅ R ₆ wherein R ₅ represents hydrogen or a methyl group, and R ₆ represents any one of a phenyl group, a glycidyl group, an acrylester group, a nitrile group, and a monovinyl-substituted phenyl group. The monomers classified into the groups A, B, and C do not prevent two or more kinds from being selected from the same group.
The second is a packing material for HPLC used for separating proteins.
The present invention will be described based on examples, but the present invention is not limited to these examples.

[0013]

Example 1 A 1-liter separable flask equipped with a cooler, a stirrer, a heating device, a thermometer, and a gas inlet tube was charged with 4
500 ml by weight of a polyvinyl alcohol aqueous solution,
A mixture of 20 g of hydroxyethyl methacrylate, 50 g of diethylene glycol dimethacrylate, 30 g of glycidyl methacrylate, 90 g of toluene and 2 g of benzoyl peroxide was supplied. Next, while blowing in nitrogen gas, the temperature was raised to 80 ° C. at a stirring speed of 300 rpm, a polymerization reaction was performed for 20 hours, and then cooled to room temperature. After cooling, the polysynthetic product is solid-liquid separated, washed repeatedly with hot water to remove residual polyvinyl alcohol, and then washed with acetone to obtain spherical particles having a particle diameter of 5 to 30 μm. Was. Finally, fine particles and coarse particles were removed from the particles to obtain a filler for HPLC having a particle size in the range of 8 to 20 μm.

[0014]

Example 2 From porous particles prepared in exactly the same manner as in Example 1 except that acrylamide was used instead of 2-hydroxyethyl methacrylate, and tetramethylol methane triacrylate was used instead of diethylene glycol dimethacrylate, fine particles and The coarse particles were removed to obtain a filler for HPLC having a particle size in the range of 8 μm to 20 μm.

[0015]

Example 3 The filler obtained in Example 1 was dispersed in 700 ml of ion-exchanged water, and 7 m of dispersed particles were placed in a stainless steel column having an inner diameter of 5 mm and a length of 30 mm by a high-pressure pump.
Filled by pumping at 1 / min. The obtained packed column (deproteinized column) was connected to a column thermostat of an HPLC device (trade name: Hewlett Packard HP-1100). A 6-way switching valve was provided between the packed column and the piping of the analytical column (ODS), and separate mobile phases were fed through the flow path. Rat serum (protein concentration: 7.
0%) as a sample, and spiked with 18 μg / ml of theophylline as a sample. 20 μl of this sample was injected into the deproteinization column, and after 2 minutes, the switching valve and the analysis column were connected, and the theophylline held in the packed column by backflushing was removed. It was eluted on an analytical column and analyzed. HPLC analysis conditions are shown below. High-performance liquid chromatograph operating conditions Column temperature bath temperature: 40 ° C Mobile phase for packed column (protein-free column): 0.02 mmol
1 phosphate buffer (pH = 7.0) mobile phase for analytical column: 0.02 mmol phosphate buffer (pH = 7.0) / CH ₃ CN = 95/5 analytical column: ODS (trade name: Hewlett Packard) Hypersil) Mobile phase flow rate: 1 ml / min Detector: diode array detector (254 nm) Further, the recovery rate of the protein recovered from the packed column (deproteinized column) was determined by the following method. After the sample was injected into the deproteinization column, the eluate was collected for 2 minutes from the outlet of the pipe downstream of the deproteinization column. Take 100 μl from this aliquot,
The protein in the eluate was quantified by the Holin method. The recovery rates of theophylline and protein were as follows. Theophrin recovery rate: 100.0% Protein recovery rate: 100.2%

[0016]

Example 4 The same sample as in Example 3 was subjected to the same method as in Example 3 using the same protein as in Example 3, except that the filler obtained in Example 2 was prepared in the same manner as in Example 3. Recovery was measured. The results are as follows. Theophrin recovery rate: 100.0% Protein recovery rate: 100.5%

[0017]

EFFECT OF THE INVENTION By using the novel filler for HPLC obtained by the present invention, the drug in the biological sample can be converted to HPC
In the case of analysis by the L method, it is possible to directly inject a sample into the HPCL apparatus for analysis, and a pretreatment operation such as a deproteinization operation or an extraction operation is not required, and the analysis time is greatly reduced.
In addition, the use of an organic solvent that may cause environmental pollution is not required.

Claims (1)

[Claims] 1. A method for dissolving two or more types of ethylenically unsaturated monomers selected from the following three types of ethylenically unsaturated monomers comprising A, B and C A packing material for liquid chromatography used for separating proteins, comprising a porous high molecular weight polymer obtained by aqueous suspension polymerization in the presence of an organic solvent in which the union does not dissolve. A CH ₂ = CR ₁ R ₂ wherein R ₁ represents hydrogen or a methyl group, and R ₂ represents either a 2-hydroxyethyl group or an amide group. Here, R ₃ and R ₄ represent hydrogen or a methyl group, and n represents an integer of 1 to 20. C CH ₂ ＣＨCHR ₅ R ₆ wherein R ₅ represents hydrogen or a methyl group, and R ₆ represents any one of a phenyl group, a glycidyl group, an acrylester group, a nitrile group, and a monovinyl-substituted phenyl group. The monomers classified into the groups A, B, and C do not prevent two or more kinds from being selected from the same group.