JPS6070353A - Hydrophobic carrier for chromatography - Google Patents

Abstract

PURPOSE:To obtain a hydrophobic carrier for high performance chromatography excellent in strength and separative recovery capacity, by using a spherical semi- hard crosslinked polymer particle containing a structural unit having a hydrophilic polyether side chain in a specific ratio or more and providing a macroporous diameter upon swelling in an aqueous solution. CONSTITUTION:A spherical semi-hard crosslinked polymer particle, which contains 50wt% or more of a structural unit having a hydrophilic polyether branch represented by formula wherein R is 1-10C aliphatic alkyl, phenyl or cyclohexyl, l and m are 1 or more, n is 0 or 1 or more and m+n is 2 or more) and provides a macroporous diameter upon swelling in an aqueous solution, is used as a carrier for chromatography. By this carrier, adsorbing capacity and an elution recovery rate can be enhanced in separative purification of living body high-molecular substances, especially, such as protein or nucleic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The invention is based on the general formula A
2-0-RCH (wherein R in the formula represents an aliphatic alkyl residue having 1 to 10 carbon atoms, a phenyl group or a cyclohexyl group,
A spherical porous semi-rigid material containing a structural unit having a hydrophilic polyether branch, where t and m are integers of 1 or more, n is 0 or an integer of 1 or more, and m+n is 2 or more. The present invention relates to a hydrophobic chromatography carrier that is a crosslinked polymer particle. Especially proteins and peptides.

The present invention relates to a separation carrier suitable for separation and purification of biopolymers such as nucleic acids through hydrophobic interaction.

Recently, separation and purification of proteins and the like by reverse phase partition chromatography or hydrophobic chromatography has become popular.

Currently, the main separation carriers used are those made by chemically bonding alkylsilane to silica gel, which have excellent mechanical strength, can use microparticles at high speeds and under high pressure, and have fast speeds or good separation performance. (Also, it has the characteristic that it exhibits almost no swelling and shrinking property due to changes in solvent composition.

However, such carriers have major problems, such as being unable to be used in a high pH range because silica gel dissolves in alkaline conditions, and generally having too strong hydrophobic adsorption power resulting in a poor recovery rate of enzyme activity. On the other hand, there are polysaccharide-based separation agents such as agarose gels with alkyl groups introduced, but their mechanical strength is extremely low, making it impossible to use microparticles at high speeds, requiring a long time to operate, and separating them. It also has disadvantages such as not being good.

In addition, porous semi-rigid cross-linked polystyrene gel, which has recently been frequently used in high-speed gel permeation chromatography, may be used, but its adsorption power is too strong and may denature proteins, and it may be susceptible to changes in solvent composition. However, it has the disadvantage of exhibiting remarkable swelling and shrinking properties, and is therefore impractical.

The inventors of the present invention have conducted extensive research into the development of an excellent hydrophobic chromatography separation carrier that can overcome the drawbacks of these conventional products, and as a result, have arrived at this invention.

That is, the present invention provides general formula A CH.

10■, -alcoomoOH, -CH, -0〒0H2-CH-CI(,
-0兑ROH (However, R in the formula represents an aliphatic alkyl residue having 1 to 10 carbon atoms, a phenyl group, or a cyclohexyl group,
t, m represent an integer of 1 or more, n represents an integer of 0 or 1 or more, m + n is 2 or more) containing at least 50 coul% or more of a structural unit having hydrophilic polyether branches. The present invention provides a hydrophobic chromatography carrier which is a spherical semi-rigid cross-linked polymer particle which swells in a water bath and has a large pore size.

The present invention will be explained in detail below.

Non-invention is general formula A Oh.

■ OH (wherein R in the formula represents an aliphatic alkyl residue having 1 to 10 carbon atoms, a phenyl group or a cyclohexyl group,
t, m are integers of 1 or more, and n is O or 1 or more,
(-cH, -0H2-otocu, -0H-(1!H,
-o4OH, which has a hydrophilic branch, a hydrophobic group is bonded to its terminal by an ether bond, and contains at least 50% or more of a structural unit having a hydrophilic polyether branch, and the terminal The hydrophobic group is designed to contact the sample and directly participate in the separation, and the hydrophilic part of the branch covers the strong hydrophobicity around the polymer main chain and crosslinking points, so that the sample such as protein can be directly separated from the polymer main chain. It is designed so that it does not come into direct contact with the chains, and it greatly contributes to the excellent protein recovery rate and enzyme activity recovery rate of the separation carrier of the present invention.

For this purpose, m+n in general formula A must be 2 or more. In addition, this hydrophilic portion improves the swelling property of the separation carrier in an aqueous solution and contributes to non-swelling and shrinking property against changes in solvent composition. R is a hydrophobic group selected from an alkyl group having 1 to 10 carbon atoms, a phenyl group, a cyclohexyl group, etc., and is directly involved in the separation.

The adsorption force tends to increase as the size of this group increases; however, if it becomes too large, the ability to remove the sample deteriorates, which is undesirable.

Specific examples of the alkyl group having 1 to 11 carbon atoms include methyl, ethyl, n-propyl, 1. so-propyl, n-butyl, 2-methylglobil, 1-methylglobil, n-amyl, 6-methylpentyl, n-hexyl,
1,3-dimethylbutyl, 3=ethylbutyl, n-heptyl, 1-methylhexyl, n-oni9f+1-merheftyl, 45.5-trinoflhexyl, diisobutylmethyl, n-decyl, etc. can be mentioned.

The separation carrier of the present invention has an average particle diameter of several microns to several tens of microns.
It is a porous particle that is a spherical particle of 0 micron and has giant pores with an average pore diameter of several 10x to several 100x in an aqueous solvent. In addition, from a few kg/C1n” to several 100/
It has a pressure resistance of Cm'' and has mechanical strength sufficient to be used as a separation carrier for high performance liquid chromatography.It also has excellent chemical stability such as alkali resistance and good durability.

Furthermore, because it is adjusted to have appropriate hydrophobicity, it can be used under relatively mild conditions without causing denaturation of proteins, etc., and the recovery rate of physiologically active substances is also good.
It can be said that this is an extremely excellent separation carrier that overcomes the drawbacks of conventionally used separation carriers.

Separation carriers obtained in the present invention include proteins and enzymes.

It can be widely used for analysis and separation and purification of peptides, nuclear pupils, etc.

The separation carrier of the present invention is produced, for example, by a crosslinking polymerization reaction of corresponding monomers or a reaction of introducing an alkyl group into the separation carrier, which serves as a base material having a hydroxyl group. First, a method using a crosslinking polymerization reaction of corresponding monomers will be explained.

In this method, a monomer, a crosslinking agent, and a polymerization initiator are dissolved in an appropriate organic solvent, and added to an aqueous solution containing a turbidity stabilizer while stirring, and the monomer and crosslinking agent are crosslinked to perform a crosslinking polymerization reaction at a temperature of 40 to 90°C. This is a method in which the agent is polymerized using a known oil-in-water type suspension polymerization reaction.

Equivalent examples include addition reaction of ethylene oxide or glycidol to the hydroxyl group of 2-hydroxyethyl methacrylate and then addition reaction of glycidyl alkyl ether, or addition reaction of glycidyl alkyl ether to polyethylene glycol monomethacrylate. Various embodiments can be mentioned, such as being able to be synthesized by

Any commonly used crosslinking agent may be used, but di- or trimethacrylates of polyols such as ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, glycerin dimethacrylate, and pentaerythritol dimethacrylate can be used as the crosslinking agent. is preferred.

In particular, crosslinking agents such as pentaerythritol dimethacrylate, which have hydroxyl groups themselves and are highly hydrophilic, are preferred.

The usage month of the crosslinking agent is 005 to 0 based on the weight of the monomer.
A range of 7 is common, preferably a range of 01 to 0.45.

iJj polymerization initiator The initiator is a regular cidical polymerization initiator.
9! can be used. Particularly preferred are benzoyl peroxide, azobisisobutyronitrile, 1-butyl barhybalate, and the like.

Use of a polymerization initiator (1 is preferably 01 to 10% by weight, particularly 0.5 to 5% by weight, based on the total of monomer and crosslinking agent).

As the slave solvent, those commonly used can be used. For example, aliphatic alcohols.

Aliphatic ketones, aliphatic esters, alicyclic alcohols, halogenated hydrocarbons, etc. that do not mix freely with water can be used.

The amount of organic solvent used is a based on the total of monomer and crosslinking agent.
A range of 5 to 5 times the volume, particularly 0.8 to -0 times the volume is preferred.

As the suspension stabilizer, commonly used ones such as polyvinyl alcohol and polyvinylpyrrolidone can be used. The amount used is in the range of 1 to 10% by weight, preferably 0.5 to 5% by weight of Q, based on water.

Next, a method for introducing an alkyl group into crosslinked polymer particles synthesized in advance will be explained.

First, it is known that spherical porous particles having the following structural units can be obtained by oil-in-water suspension polymerization of 2-hydroxyethyl methacrylate and a polyfunctional vinyl monomer (Japanese Patent Publication No. 47-21580). Public notice 2% 1977
-64187).

CH.

■ (-011,C! )5 Coo-0H2-C! H, -OH The following general formula B or Porous particles having a structural unit of general formula C are obtained.

C) (, general formula B +ar■2-a glaze (k≧2) coo+ca2-C!H2-0funH OH3 general formula C OH By further adding glycidyl alkyl ether to these porous particles, the present invention Various embodiments can be mentioned, such as being able to synthesize a target separation carrier.

Recently, the present inventors proposed that leaf-like porous particles having structural units of the general formula B or C can be synthesized directly from the corresponding monomers by an oil-in-water type suspension polymerization reaction (%
Naniaki).

For example, in a solvent system consisting of an aqueous solution containing a neutral salt and an alcohol having 4 to 6 carbon atoms, polyethylene glycol monomethacrylate is subjected to an oil-in-water suspension polymerization reaction with a crosslinking agent, and the structural unit of general formula B is It is possible to synthesize spherical porous particles having . By subjecting the branch ends of these particles to an addition reaction with glycidyl alkyl ether, or by reacting the branch ends with shrimp chlorohydrin in an aqueous solution of caustic soda, a glycidyl group is introduced, followed by an addition reaction with alcohol or phenol. The object of the present invention can be obtained.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 6-thioxer 9-7 enoxy 8-hydroxynonyl methacrylate 7507. Glycerin dimethacrylate 250? , tert-butelperpiha L/)
20 fon-phthalic alcohol was dissolved in 150 Oml, and this solution was mixed with 10 A' of an aqueous solution at 60°C in which IJaCt 300 y and polyvinyl alcohol 3002 were dissolved.
The mixture was added to the solution while stirring to form an oil-in-water type turbid state, and a cross-linking polymerization reaction was carried out at 60° C. for 16 hours.

Spherical polymer particles (general formula A, m=2, H=1°R:
(represented by a phenyl group) was obtained, so it was thoroughly washed with hot water through a glass filter, and then sieved for 20 minutes.
Particles from micron to 40 micron were collected.

The leaked particles were packed into a stainless steel tube with a diameter of 717.5 mm and a length of 50 cm, set in a liquid chromatography device, and subjected to the measurement of standard polyethylene oxide and standard polyethylene glycol under the following measurement conditions. The relationship between the logarithm of molecular weight and elution volume loss was determined.

Equipment: HLC! -802A (manufactured by Toyo Soda Co., Ltd.) Solvent: Distilled water Sample: Standard polyethylene oxide (manufactured by Toyo Soda Co., Ltd.) Standard polyethylene glycol (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) Detection: As a result of differential refractometer measurement, the exclusion limit molecular weight of polyethylene oxide is: It was confirmed that the particle size was approximately 250,000, and that the particles were porous particles having sufficiently large pores.

As a result of measuring the protein adsorption 'ltl-M of this polymer particle in an aqueous ammonium sulfate solution, t 5 M /
At the ammonium sulfate concentration of Z, good adsorption performance was shown as shown below.

α-chymotrypsinogen A 26m97ml-gel
α-chymotrypsis 7 17.5 rng/ml-gel Lysozyme 12.0 m 9 Anl-ge 1 Next, the polymer particles were packed into a column with an inner diameter of 7.5 mm and a length of 7.5 cm, and equilibrated with a 1.5 M ammonium sulfate aqueous solution. ,
A protein solution was injected and adsorbed onto the column. 1 solvent
The recovery rate was determined by eluting the protein from the hydrolyzate using a 1M ammonium sulfate aqueous solution, and as a result, a good recovery rate was obtained as shown below.

α-Chymotrypsinogen A 98% α-Chymotrypsin ion% Lysozyme 92% Example 2 Mixture of polyethylene glycol monomethacrylate (
7sor, pentaerythritol dimethacrylate 250? , tert-Petit Hahi Halle)
20 tons-7 mil alcohol 1500 m (! dissolved in oil-in-water type liε as in Example 1)! fl
An rq reaction was carried out to obtain spherical porous polymer particles.

The exclusion limit molecule m for polyethylene oxide of the obtained particles was measured in the same manner as in Example 1. As a result, the exclusion limit molecular weight was approximately 50,000.

The particles were thoroughly washed with dimethylformamide, and 10rty of the suction filter cake was washed with 200ml of dimethylformamide.
Phenyl glycidyl ether 5 was suspended in rILl.
70 at 70℃ by adding 0p and 5t of potassium carbonate.
The mixture was heated and stirred for hours.

After the reaction, the obtained polymer particles (m=2 to 10 in general formula A)
．． n==l, R: phenyl group) was thoroughly washed with dimethylformamide and distilled water, and then Example 1
The protein adsorption capacity and recovery rate were measured in the same manner as in Example 1, and excellent results equivalent to those in Example 1 were obtained.

Example 3 2-Hydroxyethyl methacrylate 800y.

Ethylene glycol dimethacrylate 200y.

Dissolve 2 oz of tert-butyl perpivalate in 1 soomz of n-amyl alcohol, and add this solution to Rh0t1.
00 F Topolyvinyl Alcohol 4DO? The mixture was added to 7 kJM aqueous solution 107 at 60° C. with stirring to form an oil-in-water suspension, and a cross-linking polymerization reaction was carried out at 60° C. for 16 hours.

The obtained polymer particles (in general formula A, m=1.n=1 to 10
．． (represented by R-nibutyl group) was treated in the same manner as in Example 1, and the exclusion limit molecular agent of polyethylene oxide was measured, and the result was approximately 600,000.

After thoroughly washing the polymer particles with dioxane, 100v of the suction P and IM cake was suspended in 100ml of dioxane, NaOH29 and glycidol 502 were added, and the mixture was stirred at 70°C for 4 hours to form the polymer particles. An addition reaction of glycidol was carried out.

After the reaction, the polymer particles were thoroughly washed with dioxane, and then
Wash thoroughly with warm water. According to the analytical value of the glycitol oligomer in the dioxane solution, about 52% of glycidol was added to the polymer particles.

The obtained polymer particles were thoroughly washed with dimethylformamide, and the suction-filtered cake 1009 was suspended in 2 [10 ml] of dimethylformamide, 502 butyl glycidyl ether and 5 ml of potassium carbonate were added, and the mixture was heated to 70°C. The mixture was heated and stirred for 10 hours.

After the reaction, the obtained polymer particles (in general formula A, m-1゜n=
1-10. After thoroughly washing the protein (represented by R-nibutyl group) with dimethylformamide and warm water, the protein adsorption capacity and recovery rate were measured in the same manner as in Example 1, and the following good results were obtained.

Protein Adsorption Capacity Recovery Rate Myogloby7 1 A37'l/al-gel 85.
0% egg alpumi y 2 L7tng7fnl-gel
97.0% α-chymotrypsin 24.5m9,4-ge
l 91 a% Example 4 The glycidol adduct of crosslinked polymer particles of 2-hydroxyethyl methacrylate obtained in the middle of Example 6 was thoroughly washed with water, and the suction-filtered cake 1002 was dissolved in distilled water 90%.
Suspend m1K, heat to 40°GK while stirring,
Ebikuro. 100 V of hydrin was added, and 1ON aqueous sodium hydroxide solution saml was added dropwise over 3 hours to convert the branch terminal hydroxyl groups of the polymer particles into glycidyl ether. After the reaction,
After thoroughly washing the polymer particles with water and dioxane, a portion of the particles was taken and hydrogen chloride was added in dioxane to quantify the produced glycidyl group. As a result, 0.51 mm o
The presence of a glycidyl group in vfn6- (x, θ1) was observed.

A dioxane-washed cake 202 of the polymer particles was suspended in 40 Inl of dioxane, and 20 Inl of n-butyl alcohol was added.
Add rnl and boron trifluoride etherate α2ml to 60
The mixture was stirred at a temperature of 4 hours to carry out an addition reaction of n-butyl alcohol to the glycidyl group.

The obtained polymer particles (in general formula A, m=1.n=i~15
．． After purifying the protein (represented by R-nibutyl group) with dioxane and water, the protein adsorption capacity and recovery rate were measured in the same manner as in Example 1, and the following results were obtained.

Protein Adsorption Capacity Recovery Rate α-Chymotrypsinogen A 22.4 Nagishika J-ge
l 99% α-chymotrypsin 14.5 mq/mlt-
gel 00% lysozyme y, srz%nd-ge
l 96% Next, the above-mentioned addition reaction of alcohol to the glycidyl group was carried out using various alcohols in place of n-butyl alcohol, and the protein content of the obtained polymer particles was
As a result of comparing the adsorption capacity h4 and the recovery rate, it was found that the protein adsorption capacity decreases as the alkyl group becomes smaller, as shown below, and that the recovery rate is excellent in both cases.

Ayayo used Protein adsorption capacity Recovery rate Patent applicant
Toyo Soda Kogyo Co., Ltd.

Claims (1)

[Claims] 1. General formula A CH5 coo4cn2-OH2-o+-;+cu2-OH-O
H2-0IRCH (wherein R in the formula represents an aliphatic argyl residue having 1 to 10 carbon atoms, a phenyl group or a cyclohexyl group,
Contains at least 50% by weight or more of a structural unit having a hydrophilic polyether branch, where t and m are integers of 1 or more, n is 0 or an integer of 1 or more, and m+n is 2 or more. , a hydrophobic chromatography carrier which is a spherical semi-rigid crosslinked polymer particle having a huge pore size when swollen in an aqueous solution.