JPS62193646A - Separating agent - Google Patents

Abstract

PURPOSE:To obtain the titled excellent separating agent for hydrophobic chromatography by polymerizing an acrylic ester compd. modified with a specified fluorine-contg. compd. and a specified hydrophilic compd. CONSTITUTION:In the separating agent for a biological substance such as proteins and enzymes, the separating agent composed of a cross-linking agent consisting of the poly(meth)acrylic ester of a polyhydric alcohol, the fluorine- contg. ester component shown by the general formula I (where R<1> is H or CH3 and R1f is a 4-17C fluoroalkyl, etc.), and the ester component of a glycerin derivative shown by the general formula II (where R<3> is H or CH3, and R<4> is H or a hydrophilic group) is used. The separating agent has high adsorptive capability on a biological substance, and the biological substance can be separated and purified in a highly active stage, since the agent can be eluted under mild conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a separating agent used when separating and purifying a target component from a mixed solution containing various components.
For details, see Proteins and Enzymes.

The present invention relates to a separation agent suitable for separating and producing biological substances such as carbohydrates and antibiotics.

[Conventional technology]

Conventionally, separation agents based on dextran or agarose gel have attracted attention in the field of chromatography for the separation and purification of biologically related substances. However, the above separation agents did not have sufficient mechanical strength.

Gel filtration chromatography as an important separation mode in the field of chromatography.

Ion exchange chromatography, affinity chromatography and hydrophobic chromatography are known. Among these, hydrophobic lithography, which utilizes the difference in hydrophobicity of protein tubes, enzymes, etc., has recently begun to be used.

This is a method for separating substances based on the difference in interaction between the hydrophobic sites of a separating agent and the hydrophobic sites of proteins, enzymes, etc. As such a separating agent for hydrophobic chromatography, a separating agent in which a linear alkyl group or an aromatic compound is bonded to the above-mentioned hydrophilic carrier is known.

Separation of the target substance was not done by light, and even the recovery rate of 1 trillion was not satisfactory.

In view of the above-mentioned circumstances, the present inventors first filed a patent application in 1986-7'.
l'IO! In this paper, we proposed a separation agent that has high mechanical strength and less nonspecific adsorption of proteins, enzymes, etc., by introducing a compound containing a specific fluorine atom into a hydrophilic carrier.

[Problem that the invention seeks to solve]

In particular, recently there has been a demand for separation agents that are effective in separating biologically related substances such as proteins and enzymes, and in particular for hydrophobic chromatography, which has both superior separation ability and optical mechanical strength.

[Means for solving problems]

As a result of repeated studies in view of one or more points, the present inventors found that a polymer obtained from an acrylic ester compound modified with a 1% fluorine-containing compound and a specific hydrophilic compound is one of the separating agents. The present invention was achieved by discovering that it has excellent separation ability and optical mechanical strength for use in hydrophobic chromatography.

[Structure of the invention]

The present invention comprises a crosslinking agent component consisting of poly(meth)acrylic ester of polyhydric alcohol, a fluorine-containing ester component represented by the following general formula (I) and/or the following general formula (II), and the following general formula ( if) and/or the glycerin derivative ester component represented by the following general formula (IV
) is a separating agent having a structure consisting essentially of a (poly)ethylene glycol ester component.

General formula (I) 40H2-0→ 000-C! H2-R'f General formula (II) (aH20+OH 000-0H20HOI'12-0- X-Rlf((
I), (II) in formula 11. R2 is a hydrogen atom or a methyl group, X is a methylene group or a carbonyl group, and 1G
1'), Rlf, Rlf represents a fluoroalkyl or fluoroaryl group having 1 to 17 carbon atoms.

) General formula (m) -(C horse-〇+on Coo -0H20HOH, -〇-R4 general formula) R5 (OH2-0+ ■ 000 +0H20H20%H (GU), (IV) In the formula, R3 and R5 are hydrogen atoms or methyl group R4 represents a hydrogen atom or a hydrophilic group.

n is an l-da integer. ) The present invention will be explained in detail below.

In the present invention, the term "separating agent" refers to a broad concept used in separation operations such as gel filtration chromatography, ion exchange chromatography, and hydrophobic chromatography. means.

The separating agent of the present invention has the above-mentioned general formula (I
) or the fluorine-containing ester component of (TI) and the glycerin derivative ester component of the above general formula (IIJ) or the (poly)ethylene glycol ester component of the above general formula (IV) that imparts hydrophilicity to the separating agent. It consists essentially of three components:

Polyacrylic esters or polymethacrylic esters of polyhydric alcohols constituting the crosslinking agent component include 1 usually ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and tetraethylene. Glycol dimethacrylate, propylene glycol dimethacrylate, pentaerythritol dimethacrylate, glycerol dimethacrylate, glycerol trimethacrylate and their corresponding acrylic esters are used alone or in combination. Among these, ethylene glycol or diethylene glycol dimethacrylate or glycerin dimethacrylate is preferably used.

The fluorine-containing ester component is represented by the following general formula (I) or general formula (II).

−7= ■ , +CH2−C+ ・・・・・・・・・
・・・・・・・・・・・・・・・・・・(I)Coo-
0H2-R'f (in the formula, R1, R'', Octyl group -'l'I, 2
Examples include a tetrahydro-perfluorodecyl group, a hexafluoroisoglobil group, and a linear fluoroalkyl group is particularly preferred. Examples of the fluoroaryl group include a trifluorophenyl group and a pentafluorophenyl group.

The glycerin derivative ester component has the following general formula (III
), and the (poly)ethylene glycol ester component is represented by the following general formula (IV).

+OH, -0+ ・・・・・・
・・・・・・・・・・・・(IV)Coo +0H20H2
0+nH (in the formula, RB, R4, R5, n are as described above) R of the glycerin derivative ester component represented by the general formula (m)
The simplest form of 4 is hydrogen, but hydroxy hydrocarbon groups are also preferred. For example, glycerin derivative esters are preferably used, in which a glycerin derivative ester such as glycerol, pentaerythritol, ethylene glycol, glucose, etc. is bonded to the hydroxyl group at the 3-position of glycerin via its hydroxyl group to form an ether bond.

In order to incorporate these glycerin derivative ester components into the separating agent of the present invention, (meth)acrylic acid esterified with these glycerin derivatives may be used in the polymerization reaction. ) Acrylate may be used in the polymerization reaction, and then the glycidyl groups in the polymer may be reacted with water or polyol.

In addition, the (poly)ethylene glycol ester component represented by the above general formula (fV) can be introduced into the separating agent by using, for example, a (meth)acrylic acid ester of the corresponding sulfur (poly)ethylene glycol in the polymerization reaction. I can do it. For example, if n=/, β-hydroxyethyl (
Meta)acrylate may be used.

The separating agent of the present invention essentially consists of the above-mentioned crosslinking agent component, fluorine-containing ester component, glycerin derivative ester component, and/or (poly)ethylene glycol ester component, but may contain other components as long as their functions are not impaired. May contain. For example, an alkyl ester may be contained in a separating agent by coexisting a small amount of (meth)acrylic acid alkyl ester such as methyl methacrylate during polymerization. It is advantageous for use in

The aforementioned crosslinking agent component should account for 3% or more by weight in the crosslinked copolymer. It preferably accounts for 10 to 0% by weight, particularly preferably 10% to 0% by weight. If the proportion of the crosslinking agent in the crosslinked copolymer is too small, the swelling property of the crosslinked copolymer will increase, leading to a decrease in mechanical strength. On the other hand, if it is too large, it may increase the strength of non-specific adsorption of proteins due to a decrease in the glycerin derivative ester component or (poly)ethylene glycol ester component, or decrease the interaction with the separation object due to a decrease in the fluorine-containing ester component. bring.

The fluorine-containing ester component has a content of fluorine atoms in one separating agent of 0. It is preferable to make it exist so that it is within the range of /~JOM view section. Fluorine atom content is 0.1% by weight
When the content is smaller, it is less practical as a separating agent, and when the content is greater than 30% by weight, various problems occur, such as a decrease in hydrophilicity and a decrease in separation ability and recovery amount. A more preferable fluorine atom content is /-/j 11-h, and when it is within this range, it exhibits better performance as a separating agent for hydrophobic chromatography. Further, the glycerin derivative ester component and/or the (poly)ethylene glycol ester component is a glycerin derivative ester component.

The molar fraction relative to the sum of the moles of the (poly)ethylene glycol esterone component and the fluorine-containing ester component is
Preferably, it is present in the copolymer in such a proportion that it accounts for 99.9% of A'0-99.9%. If the molar content z of the glycerin derivative ester component or the (poly)ethylene glyco-nonester component is low, the hydrophilicity of the separating agent decreases.

Next, a method for producing the separating agent of the present invention will be explained.

One example of the method for producing the separating agent of the present invention is as follows.

A (meth)acrylic ester that provides a fluorine-containing ester component represented by general formula (I) or (II), and a glycerin derivative ester component or (poly)ethylene glycol ester component represented by general formula (IIIT) or (IV). (meth)acrylic ester giving
A method for producing a separating agent containing each component in one step by copolymerizing it with a crosslinking agent such as ethylene glycol dimethac+)V-) can be mentioned. The (meth)acrylic acid ester that provides the fluorine-containing ester component used in copolymerization is /, l
Dihydro-perfluorooctyl (meth)acrylate, perfluorohebutylcarbonylglyceryl (meth)
Acrylic), /, /.

! ; ToU? −)” Roperfluoropentyl < mep
) acrylate, etc.

Another method for producing the separating agent of the present invention is glycidyl (
Examples include a method in which meth)acrylate and a crosslinking agent such as ethylene glycol dimethacrylate are copolymerized, and then the glycidyl groups of the copolymer are chemically modified with a fluorine-containing compound and a polyol. Fluorine compounds used for chemical modification include /, /dihydroperfluorooctane-
l-ol, t, y, s trihydrono-fluorobentane-no-ol, no(-fluorooctaneff,
/, /, cotrihydro) -fluoro/-decene, pentafluorophenoxyacetic acid.

Examples include pentafluoroacetic acid, pentafluorobenzyl alcohol, tetrafluorophenol, /-(pentafluorophenyl)ethanol, and the like. Examples of polyols include glycerol, pentaerythritol, ethylene glycol, and glucose. Note that the epoxy group may be ring-opened by reacting water instead of the polyol.

Still another method for producing the separating agent of the present invention is a combination of the above-mentioned one production method, which is a (meth)acrylic Jvill ester that provides a fluorine-containing ester component represented by the general formula (I) or (II), and any (meth)acrylic ester selected from (meth)acrylic esters and glycidi/I/(meth). ) This is a method in which acrylate is copolymerized with a crosslinking agent, and then the glycidyl groups of the copolymer are chemically modified with a fluorine-containing compound, polyol, water, or the like.

In any of the production methods, the polymerization reaction can be carried out in a known manner, but it is usually carried out in suspension polymerization in an aqueous medium containing an appropriate dispersion stabilizer to obtain a spherical crosslinked copolymer. preferable.

Dispersion stabilizers typically include gelatin, sodium polyacrylate, and carboxymethyl cellulose.

Polyvinyl alcohol or the like is used. Further, it is preferable to dissolve salts in the aqueous medium to prevent the polymerizable monomer from dissolving in the aqueous medium.

As the salts, sodium chloride, calcium chloride, sodium sulfate, etc. are used. As a polymerization initiator, benzoyl peroxide, t-butylhydrocarbon oxide,
Azobisisobutyronitrile, dimethylazobis(methylvalate).

Azobis (α,α-dimethylvaleronitrile).

Organic peroxides and organic azobis compounds such as λ, coazobisuco, and tadimethylvaleronitrile are used.

These polymerization initiators usually have θ of 1 reaction raw material, θ/~3
Used in quantities that are weighted.

The polymerization reaction is usually completed in t to -0 hours at 5o-qoC under stirring. Further, in this polymerization reaction, a porous crosslinked copolymer can be obtained by employing the following known method. Namely, (1) a method in which polymerization is carried out in the presence of a solvent that is homogeneously mixed with the reaction raw material but has low affinity for the crosslinked copolymer to be produced. (2) A method in which a linear polymer such as polystyrene is present in the reaction raw material and polymerized, and the linear polymer is extracted and removed from the resulting crosslinked polymer. (3) Crosslinks formed by polymerization in the presence of a linear polymer such as polystyrene and a solvent such as toluene, ethyl acetate, or dimethylformamide that has an affinity for the resulting crosslinked copolymer in the reaction raw materials. y5 method for extracting and removing the linear polymer from the copolymer. (4) In the reaction raw material (1
), etc., which are homogeneously mixed with the solvent and reaction raw materials mentioned above and are polymerized in the coexistence of a solvent that has an affinity for the crosslinked copolymer to be produced.

One of these methods is usually carried out by adding an organic solvent having a low affinity for the crosslinked copolymer to the raw material (1/16 times) and carrying out suspension polymerization according to the method (1) above. The organic solvent is not particularly limited as long as it is inert to glycidyl groups and hydroxide groups, but usually includes benzene, toluene, ethylbenzene, chlorobenzene, n-octane, and cyclohexanol.

Butyl ether, acyl ether, butyl vinegar.

Cyclohexanone or the like is used, and the amount used is usually 0.03 to 3 times the weight of all monomer components.

After thorough washing, the separation agent obtained in this way is 4. r
Used as a separation agent for seed applications.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to the following Examples unless the gist thereof is exceeded.

Example 1 Glycidyl methacrylate; tig, diethylene glycol dimethacrylate gg, cyclohexanol-
1 (mixture of # and his, λ'-asohysuko, 0.3 g of l-dimethylvaleronitrile, ion exchange water, 2 S
Add a knife to a solution of polyvinyl alcohol 7, gg, and sodium chloride dissolved in 0ml, and add 8g while stirring.
Suspension polymerization was carried out at 0°C for 8 hours. After cooling the reactant, the generated copolymer particles were separated and washed with water. Next, this copolymer was washed once each with methanol and acetone, and then subjected to light separation and water washing to obtain a white porous resin with an average particle size of θ μm (this resin is a methacrylic acid-based crosslinked copolymer (referred to as A).

Polymerization convergence was 94%.

This methacrylic acid-based crosslinked copolymer p, iog and perfluorooctanoic acid i, Ag were placed in a flask, and added to l, terdioxane, and 01n! ! The mixture was heated to 1-0° C. using a flame, and the reaction was carried out for 6 hours. Furthermore, 207 nl of a 70% sulfuric acid aqueous solution was added into this, and a reaction was carried out at 30° C. for S hours. After cooling, the reaction mixture was separated and thoroughly washed with acetone and water.

A separating agent was obtained. The elemental analysis value of this separation agent was first order.

Elemental analysis value c: 33. i % H: 7.0% F: /, si% In addition, in the infrared absorption spectrum, an absorption band derived from methacrylic acid ester was observed at /, 7rio CTL-1°/, 7g OcIrL-''.

The ratio of the fluorine-containing ester component, the hydrophilic ester component, and the crosslinking agent component of this separating agent is 1 molar ratio of 1:1:
g:/ri (each J, ?, 2..2.twt% by weight)
)Met.

Example 1: 10 g of methacrylic acid-based crosslinked co-N coalescence A produced in FJ/7. /dihydroperfluorooctane-/-ol, 2. 7. Put JjJ into the flask and put it inside. Tardioxane! ; Oml and 0.6μ of sodium hydride were mixed, and a reaction was carried out under heat at 30° C. for an hour. The following reaction was carried out in the same manner as in Example 1 to obtain a separating agent. The elemental analysis values of this separation agent were as follows.

Elemental analysis value C: 32.3% H: A, 1g% F:,! ! -, 7% The proportions of the fluorine-containing ester component, hydrophilic ester component, and crosslinking agent component of this separation agent are 1 molar ratio, respectively:
:lO:II, 8 (weight percentage each //, A O, jJwt
%) and ivy.

Implementation f113 Methacrylic acid-based crosslinked copolymer A1 produced in Example 1
0g and pentafluorophenoxyacetic acid s, og were placed in a flask, 50 ml of terdioxane was put into this, and concentrated sulfuric acid sulfuric acid, RG was heated to 80°C and a reaction was carried out for s hours. Ta. The following reaction was carried out in the same manner as in Example 1 to obtain a separating agent. The elemental analysis values of this separation agent were as follows.

Elemental analysis value C: sa, 7% H near, flat F: g, 7% The proportions of the fluorine-containing ester component, hydrophilic ester component, and crosslinking agent component of this separation agent are 1 molar ratio, respectively:
? l:/, q (weight ratio of each/g,, tq, :1
．． 7 wt%).

Example G Example/methacrylic acid-based crosslinked copolymer Al produced in
0g and 7. /Dihydroperfluorooctane-/-ol x, py was placed in a flask and dried/, l
I-dioxane tismz was added, and metal sodium o and isg were added, and the mixture was gradually heated to carry out a reaction at about 83°C for an hour.

After the reaction, the solution was filtered, dried and washed with dioxane. Next, fill the flask with polyethylene again,
i,y-dioxane 41! ;rrd! , ethylene glycol3. og and add 0.0 ml of concentrated sulfuric acid into it. The left part of the mixture was added dropwise, and the reaction was carried out at 0°C for 3 hours. After the reaction was completed, the polymer was filtered and washed with light and water to remove dioxane and ethylene glycol to obtain a separating agent. The elemental analysis values of this separation agent were as follows.

Elemental analysis value C: left 1.3% H: Otsu, 7% F: +2i% The proportions of the fluorine-containing ester component, hydrophilic ester component, and crosslinking agent component of this separation agent are 1 molar ratio, respectively.
:lIo:q, sc heavy state ratio each 3,74.2/wt%
)Met.

Example S The same operation as in Example 1 was carried out, except that 3.0 g of glycerol was used as the hydrophilic compound instead of ethylene glycol J, 09. The elemental analysis values of the obtained separation agent were as follows.

Elemental analysis value C: left /, J % H, A, mouth F: 2.3% The proportions of the fluorine-containing ester component, hydrophilic ester component, and crosslinking agent component of this separation agent are 1 molar ratio, respectively /:
lI/:q,,y(each in weight proportion,!ts'/Ar,,
2θwt%).

Example 6 Glycidyl methacrylate 3. ig, ethylene glycol dimethacrylate/, ll/i, /, /dihydroperfluorooctyl methacrylate o, scoy,
A mixture of 11.79 g of cyclohexanol was added to a solution in which 3 g of polyvinyl alcohol o, t and 0.97 fi of sodium chloride were dissolved in JOml of ion-exchanged water, and suspension polymerization was carried out at goC for 8 hours with stirring. After cooling the reaction product, the produced copolymer particles were separated and washed with water. Next, this copolymer was mixed with methanol and acetonthene, respectively. 2
Washed several times, further washed with light and water, and the average particle size was 10.
A white porous resin of S μm was obtained. The polymerization rate was 93%.

This methacrylic acid copolymer s, og was placed in a flask, and 10 ml of a 3% sulfuric acid aqueous solution was poured into the flask.

The reaction was carried out at 30°C for S hours. After cooling, take the copolymer out and wash it thoroughly with acetone and water.

A separating agent was obtained. The elemental analysis values of this separation agent were as follows.

Elemental analysis value a: rl, q% Hnia, /lI% F: Fu 89% The proportions of the fluorine-containing ester component, hydrophilic ester component, and crosslinking agent component of this separation agent are 9 molar ratios, respectively:
Yu θ: 6. Da (10 each in weight percentage, Otsu x,, 211
wt%).

Example 7 - Protein adsorption/desorption test - Bovine serum γ-globulin and bovine serum albumin manufactured by Eligma were each added to a concentration corresponding to O12 weight.
0-. 0./M Tris salt buffer solution (pH 7) containing 30% by weight of ammonium sulfate at various concentrations.
, yO). -10,000, Example/, J, lI and 6 each of the separation agents obtained in s and oomt were added to 0. /OM
After sufficient equilibration with Tris' and hydrochloric acid buffer solution 'O (pHt, sO), centrifugation was performed to remove adhering water, and the solution was added to the protein solutions 1001 with different concentrations of ammonium sulfate, and incubated at io°C.
After shaking for 6 hours, the TIV absorbance of the supernatant solution (xgO
The amount of protein adsorbed onto the separation agent was determined by measuring the amount of protein adsorbed onto the separation agent.

Next, the entire amount of the separation agent that had adsorbed the protein was distributed to each household, and a 0.00-ml solution containing ammonium sulfate at the same concentration as that used in the adsorption measurement experiment was used. /M Tris-jJ acid buffer solution SO1
Washed with. After centrifuging each separation agent and removing attached water, 0. /M Trio/HCl buffer solution (pH
7,kO) in 100ynl.

It was shaken at 70°C for 6 hours. The amount of protein desorbed was determined by measuring the absorbance of the supernatant solution.

The results of measuring the amounts of bovine serum r-globulin and bovine serum albumin adsorbed and desorbed by the separation agents produced in Example 1, J, DA, and A in the above-mentioned 1-sho are shown in Tables-/-Table-4, respectively. Shown below.

The following three types of protein mixed solutions were separated under the following conditions. The obtained chromatogram is shown in FIG.

Physical properties of the protein used Protein Molecular weight (×lOli Isoelectric point Cytochrome O/Yuko IB Ribonuclease A I3.7 9
．． Ni~Deku Ovalbumin 4133
Alcohol dehydrogenase /lIOyoS~1
0 Thyroglobulin A6 1A measurement conditions Column = 7-t mx to X7-tllE flow rate:
/, 0TLI! /min Elution: A-)B Co□ min Linear gradient detection: T]V280nm Injection R: , 2apl (7.5 min for each protein/719.
(Dissolved in ON) [Effects of the Invention] Since the separating agent of the present invention essentially consists of a hydrophilic ester structure, it itself is inert to biopolymers. Because the carrier has a fluorine compound introduced into its base, it has a high adsorption capacity for biologically related substances and can be eluted under mild conditions, allowing biologically related substances such as proteins to be separated in a highly active state. It has the characteristic of being refinable.

Furthermore, the separating agent of the present invention has a high mechanical strength.

There is little pressure loss when filling the column. Therefore, when using the column method, high flow rate processing is possible even if the height of one layer is increased.
It is advantageous for purification.

Therefore, the separating agent of the present invention will greatly contribute to the field of separation of biologically related substances and nano-manufacturing, and is expected to be used as a separating agent for hydrophobic chromatography.

[Brief explanation of drawings]

FIG. 1 is a chromatogram obtained when a mixed solution containing S type proteins was separated using the separation agent produced in Example. The horizontal axis is retention time. The vertical axis indicates the absorbance at -gQnm. In the figure, each peak is /: cytochrome C1-diribonuclease AS J: ovalbumin, G: alcohol dehydrogenase, S: thyroglobulin.

Claims (3)

[Claims] (1) A crosslinking agent component consisting of poly(meth)acrylic acid ester of polyhydric alcohol, and the following general formula (I) and /
or a fluorine-containing ester component represented by the following general formula (II), a glycerin derivative ester component represented by the following general formula (III), and/or a (poly)ethylene glycol ester component represented by the following general formula (IV). Separating agent with a structure that makes it useful. General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ General formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formulas (I) and (II), R^1 and R^2 are hydrogen Atom or methyl group; ▼ General formula (IV) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (In formulas (III) and (IV), R^3 and R^5 are hydrogen atoms or methyl groups, and R^4 is hydrogen atoms or a hydrophilic group, where n is an integer of 1 to 4.) (2) R^1_f in the above general formula (I) or (II)
, R^2_f is a linear fluoroalkyl group, the separating agent according to claim 1. (3) The separating agent according to claim 1 or 2, wherein the content of fluorine atoms in the separating agent is 0.1 to 30% by weight.