JPS6274904A - Production of support for affinity chromatography - Google Patents

Abstract

PURPOSE:To obtain the title support high in mechanical strength and large in adsorption capacity of enzymes and proteins, by reacting a porous crosslinked copolymer obtained by copolymerizing a crosslinking agent component with a monomer component with a specified pigment. CONSTITUTION:A crosslinking agent component (A) selected from polyolpoly (meth)acrylates is mixed with a monomer component (B) selected from glycidyl(meth)acrylates, a pore-forming agent, a polymerization initiator, etc., and the monomers are copolymerized at 50-90 deg.C for 6-20hr to obtain a porous crosslinked copolymer (C) having a pore volume in a dry state of 0.2-2.0ml/g. After the epoxy groups of component C are hydrolyzed, it is reacted with a pigment of the formula (wherein one of R<1> and R<2> is H and the other is -SO3(-)) or component C is reacted with the pigment of the formula before the remaining epoxy groups in component C are hydrolyzed to obtain a support for affinity chromatography.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a carrier for affinity chromatography, and more specifically, a carrier that has a specific affinity for biologically related substances such as specific proteins and enzymes. This invention relates to a carrier for affinity chromatography that supports a dye and has high separation ability and mechanical strength that is comparable to sieving.

[Conventional technology]

The blue pigment represented by the following general formula (1) includes phosphofluorchitonase, protein kinase, etc. (7) A group of kinases that use ATP as a substrate, alcohol dehydrogenase,
It adsorbs various enzymes, including dehydrogenases that use NAD as a coenzyme, such as lactate dehydrogenase. This dye also has a specific affinity for non-enzymatic proteins such as albumin, interferon, blood coagulation factors and lipoproteins. Taking advantage of these properties, this blue pigment has been immobilized on water-insoluble finger bodies and used for the separation and purification of physiologically active substances such as the enzymes and proteins mentioned above.

(In the formula, one of R1 and R2 represents a hydrogen atom and the other represents a 180s group.) Conventionally, carriers for affinity chromatography on which this blue dye is immobilized include those supported on cross-linked agarose or cross-linked cellulose. However, it is generally known that adsorption capacity is low, elution of adsorbed enzymes and proteins is difficult in many cases, mechanical strength is weak, and price is high, so it is not necessarily advantageous for industrial use. It wasn't.

[Object of the Invention] In view of the above circumstances, the present invention was developed as a result of various studies aimed at developing an industrially advantageous support for affinity chromatography with a blue dye. We have completed the present invention by discovering that by introducing a dye, it is possible to obtain a carrier that has high mechanical strength, a large adsorption capacity for enzymes and proteins, and a high recovery rate.

[Structure of the invention]

That is, the gist of the present invention is that the pore volume of a copolymer obtained with a crosslinking agent component selected from polyol polyacrylate and polyol polymethacrylate and a monomer component selected from glycidyl acrylate and glycidyl methacrylate is in a dry state. After copolymerizing under conditions within the range of Oo- to λ, Od/f, (1) After hydrolyzing the epoxy groups in the obtained porous crosslinked copolymer, it is expressed by the following general formula (I). or (11) react the obtained porous crosslinked copolymer with the following general formula (1).
This invention relates to an fJ-B method for a carrier for affinity chromatography, which is characterized in that the residual epoxy groups in the porous crosslinked copolymer are hydrolyzed after reacting with a dye represented by n.

(In the formula, one of R1 and R2 represents a hydrogen atom and the other represents a 1803 group.) The present invention will be described in detail below.

The affinity chromatography body according to the present invention contains a polyol polyacrylate or a polyol polymethacrylate having a body-specific crosslinked structure, and a dye represented by the above general formula (1) that exhibits a specific interaction. It is substantially contained from the Il1 position of three structures having different functions: ester and glycerin ester represented by the following general formula (II) which imparts hydrophilicity.

(In the formula, R represents a hydrogen atom or a methyl group) As polyol polyacrylate or polyol polymethacrylate, 1 usually includes ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, propylene glycol dimethacrylate, etc. Rudime l acrylate, diglopyrene glycol dimethacrylate,
Tripropylene glycol dimethacrylate, trimethylolpronotrimethacrylate, pentaerythritol tetramethacrylate)%'13-butylene glycol dimethacrylate and their corresponding acrylates are used alone or in mixtures.

Preferably, diacrylate or dimethacrylate of ethylene glycol is used. These crosslinking agents should account for at least 1 O (fr amount) in the carrier. Preferably 7O-fO (weight)% of w-isomer, especially Jo-Jo(i
Mayuzumi) occupies Chi. If the proportion of the crosslinking agent in the carrier is too small, the carrier will have a large swelling property, resulting in a decrease in mechanical strength, making it unsuitable as a carrier for chromatography.

On the other hand, if the ratio of the crosslinking agent becomes too large, the protein adsorption capacity will decrease due to a decrease in the dye-containing ester moiety, or the hydrophilicity will decrease due to a decrease in the glycerin ester moiety.

Next, the dye-containing ester will be described. A dye represented by general formula (I) and
.

butts JG-A (C1ba-Geigy company split)
Alternatively, ProciOryDbllts H"B (KoC Kosha Momme) are commercially available and can be used. As an introduction method.

Two methods are possible.

(1) A method of hydrolyzing the epoxy groups in the crosslinked copolymer and then reacting the dye. According to this method, the dye is introduced into the hydroxyl group of the glycerin ester moiety represented by the general formula (n), and Take the structural unit shown by (II[).

CIlri (In the formula, R represents a hydrogen atom or a methyl group.

One of R1 and R2 is a hydrogen atom and the other is 1803
(11) A method of reacting a crosslinked copolymer with a dye and then hydrolyzing the remaining epoxy groups in the crosslinked copolymer. According to this method, the dye has the following general formula (to) The epoxy group of the glycidyl ester moiety represented by the following general formula (
One can be considered to be introduced as a structural unit represented by v), and the other is to be introduced as a structural unit represented by the above general formula (III).

Although it is not clear, it is presumed that the dye is probably introduced in the form of the two types mentioned above.

(In the formula, Ff represents a hydrogen atom or a methyl group) (In the formula,
(R represents a hydrogen atom or a methyl group; one of R1 and R2 represents a hydrogen atom and the other represents a -808 group) After introducing the dye using method (11), the remaining epoxy group is hydrolyzed. Therefore, flJ, (ill) Regardless of which method the dye is introduced into, the carrier according to the present invention contains a polyol polyacrylate or polyol polymethacrylate moiety that provides a crosslinked structure, an ester moiety containing a dye, and a general formula There is no change in the fact that it is substantially composed of the three structural units of the glycerin ester moiety represented by (II).

The dye-containing ester has a dye introduction t of the carrier of 0, j-30
It is present in the phase body in an amount that gives 0μno1/f. If the amount introduced is smaller than OJ μmol/f, it is not practical as a phase for affinity chromatography, and if the amount introduced is larger than 500 μmol/f, the hydrophilicity of the phase decreases, resulting in a decrease in protein recovery. It causes moderate problems.

What is the more suitable amount of dye introduced into the carrier according to the present invention? ~/
00 pmo1/9. Further, the glycerin ester units are preferably present in the carrier in such an amount that the number of moles of the glycerin ester units accounts for 11% or more of the total number of moles of (glycerin ester units and ten dye-containing ester units).

If the amount of glycerin ester is small, the hydrophilicity of the carrier will be reduced.

The phase according to the present invention consists essentially of the above-mentioned crosslinker units, dye-containing ester units and glycerin ester units, but if desired, units such as methyl or ethyl esters of acrylic acid or methacrylic acid may be added. It may be included.

The carrier according to the present invention is produced by copolymerizing a crosslinking agent with a glycidyl ester of acrylic acid or methacrylic acid to form a crosslinked copolymer, and (1) reacting the epoxy of the glycidyl ester with water to open the ring. Alternatively, by directly reacting one dye to the cross-linked copolymer, the remaining epoxy group of the glycidyl ester is reacted with water to open the ring. M can be built.

The method for making the carrier porous will be described later, but when targeting macromolecules such as proteins, it is essential to make the carrier porous. If the porosity is insufficient, the protein cannot diffuse into the pores in the carrier, and the absorption reaction occurs only on the surface of the phase particles, resulting in a decrease in absorption Mum. or,
On the other hand, if the porosity progresses too much, the mechanical strength of the phase decreases, causing problems in practical use as a cage for lithography.

The pore volume of the carrier is 0.2 to Od/? at the crosslinked copolymer stage before introducing the dye. - Should be within the range of dry copolymers, preferably.

0.11-w /,! Shoulder e/f-dried copolymer.

The polymerization reaction may be carried out by any known method, but it is usually preferable to carry out suspension polymerization in an aqueous medium in the presence of a suitable dispersion agent to obtain a particulate copolymer. As the dispersion stabilizer, gelatin, sodium polyacrylate, carboxymethyl cellulose, polyvinyl alcohol, etc. are usually used. Further, it is preferable to dissolve a salt in the aqueous medium to prevent the polymerization reaction raw material from dissolving in the aqueous medium. As the salt, sodium chloride, lucium chloride, sodium sulfate, sodium carbonate, etc. are used. The polymerization reaction is generally carried out using a suitable polymerization initiator. A polymerization initiator (7) is generally benzoyl peroxide,
1-Butyl hydroperoxide, azobisin butinide ■ nor, dimethyl azobis (meterba L/-))
, 7/His(α,a-dimethylvaleronitrile), 2.
2'-7 Zobisu 2. Organic peroxides such as II-dimethylvaleronitrile and nuazobis compounds are used. These polymerization initiators.

Usually, it is used in an odd amount such as oot-t (1% by weight) of the reaction raw material.

The polymerization reaction is usually completed in 6 to 20 hours at −90° C. with stirring. In order to obtain a porous crosslinked copolymer, a porosity-forming agent may be present in addition to the monopolymerizable monomer during the polymerization reaction, and the porosity-forming agent may be removed after one reaction.

The method for preparing porous crosslinked copolymers is well known, and is usually (
1) A method of polymerizing in the presence of a solvent that is uniformly mixed with the reaction raw material but has low affinity for the crosslinked copolymer to be produced, and then removing the solvent, (2) A method of polymerizing the reaction raw material, and then removing the solvent. (3) A method of polymerizing with a linear polymer present in the reaction material and extracting and removing the linear polymer from the produced crosslinked copolymer; A method of polymerizing in the presence of an compatible solvent and extracting and removing the linear polymer from the resulting crosslinked copolymer, (4) uniformly distributing the solvent in (1) and the reaction raw materials into the reaction raw materials. Either method is used in which the polymerization is carried out in the coexistence of a solvent that has an affinity for the crosslinked copolymer to be mixed and produced, and then the solvent is removed.

As mentioned above, the chromatography carrier according to the present invention, which targets macromolecules such as proteins, has a pore volume measured in a dry state at the crosslinked copolymer stage before introducing the blue dye. should be in the range of 02-20 rye/f, preferably O1≠~t, s r
It should be within the prl/l range.

As the above-mentioned porosity-forming agent, benzene is preferably used in the polymerization reaction according to the present invention.

Examples include toluene, ethylbenzene, rubenzene, n-octane, dichloroethane, n-octatool, cyclohexanol, butyl acetate, diptyl phthalate, methylinbutylcarbinol, etc., and the amount used is usually based on the total polymerizable monomers. It is said to be 5 to 300% (weight 'M).

The reaction between the crosslinked copolymer and the dye is carried out as follows.

(1) When the epoxy groups in the cross-linked copolymer are subjected to calohydrolysis and then reacted with the dye. Hydrolysis of the epoxy groups is carried out by diluting the cross-linked copolymer in water containing sulfuric acid. 3-2 at 30-100℃
It is sufficient to hold it for 0 hours. The glycerin monoester-containing crosslinked copolymer thus obtained is suspended in an aqueous solution containing a dye, and after stirring for 30 to 60 minutes, sodium carbonate, sodium hydroxide, etc. are added to adjust the pH of the system. The mixture may be made alkaline and reacted at 20 to 0°C for 0.5 to 12 hours. At this time, the amount of dye introduced can be increased by adding an inorganic salt such as sodium chloride or Glauber's salt. Further, the amount of dye introduced can also be adjusted by selecting the above-mentioned reaction conditions.

(11) When directly reacting a dye with a crosslinked copolymer, suspend the glycidyl ester-containing crosslinked copolymer in an aqueous solution containing an alkali such as IJ Wam and 20 to 0°C. The reaction may be carried out for an hour of O,S. At this time, the amount of dye introduced can be increased by adding an inorganic salt such as common salt or mirabilite. Further, the amount of dye introduced can also be adjusted by selecting the above-mentioned reaction conditions. Ring-opening reaction of the epoxy group in the remaining glycidyl ester part after introducing the dye?・Well, the crosslinked copolymer may be suspended in water containing sulfuric acid or phosphoric acid and held at 30 to 100°C for 20 hours.

〔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 it exceeds the gist thereof.

Example-7 Crisidyl methacrylate 2109. Ethylene glycol dimethacrylate 02. Toluene 3009 and 2.
2'-azobis-λ, ≠-dimethylvaleronitrile 3t
A mixture of demineralized water, polyvinyl alcohol-2/F in 2100 yd, and a solution P in which Hirof was dissolved in sodium chloride.
This was added, and suspension polymerization was carried out for 70'c'''c/hour while stirring. After the reaction product was cooled, the produced copolymer particles were separated and washed with water.Then, this copolymer was dissolved in methanol/
, j and stirred at room temperature for 3 hours was repeated three times, filtered, and dried at 10° C. for 16 hours.

Porous copolymer particles with an average diameter of 2111/80μ (pore volume of dry particles determined by mercury intrusion method using Autobore Octopus Model 00 manufactured by Gotsu Seisakusho: 0.75 m)
e/l) (hereinafter abbreviated as Polymer A).

) The yield was very low.

Polymer A was placed in an aqueous solution of 7 io% sulfuric acid 7j0 and kept at SO<0>C for 5 hours without stirring. After cooling, the reactant ftP was separated and thoroughly washed with water to obtain glycerin heptanoester-containing crosslinked copolymer particles j j Ord f:. (Hereinafter abbreviated as Polymer B.) Thereafter, accumulated water in Polymer B was removed using a centrifuge. Among them 'Ij? 'f:
, 9 as color dye 01bachronoblue 3 G-
A (C! iba-Geigy company'A) 0. ♂V.

Pour into 100 d of an aqueous solution containing NaC1101.

The mixture was stirred at 20°C for 1 hour. Then anhydrous Na2CO310
, tf were added, and the mixture was allowed to react at 0° C. for r hours.

After distributing the reactants from door to door, wash them thoroughly with water and give A/-! 1 product was obtained.

The absorbance at 613 nm of the unreacted dye-containing solution collected during water washing was measured, the amount of unreacted dye was determined, and the amount of dye supported by the product was calculated, and it was found to be 5.7 (2-dye/l-carrier).

Example-2 ■ Blue dye 0j-bacron'blue 3 G
-A o3! Exactly the same operation as in Example 1 was performed except that IF was used.

A product of ttrd was obtained, and the amount of one dye supported was 2J (?-dye/one carrier).

Example-3 Blue dye C! 1bacronobljte 3G-
Exactly the same operation as in Example-7 was performed except that A f O,2 was used.

A product of J/d was obtained, and the amount of dye supported was i,3 (2-dye/one carrier).

Example-≠ The same operation as in Example-7 was performed except that 2.02 of the blue dye C1bacron@blue 3 G-A was used.

A product of JJd was obtained, and the amount of dye supported was /! (f-dye/ as one carrier).

Example-j ■ Blue dye 01bacron bl+ie j G -A
θ,! v1 Anhydrous Na20032. Polymer A 3 obtained in Example-1 was added to 100 yd of an aqueous solution containing /f dissolved therein, and reacted at to°C for 3 hours. The reactants were separated and thoroughly washed with water. Next, the reactant was added to an aqueous solution of ios sulfuric acid.
ml and reacted at 0°C for 5 hours. The reactants were washed with water from door to door to obtain product/j-.

The amount of dye supported was determined in the same manner as in Example 7 and was found to be (-dye/one carrier).

Application example - A predetermined amount of bovine serum albumin (Sigma reagent) is 0. /
M) Aldavan solution H at various concentrations (0.06 W/'J% to 0.70 □%) was prepared by dissolving it in a lithium-hydrochloric acid buffer solution (pH 7, jO).

Example-/, 2. Each of the dye-supporting carriers obtained in J and F,
oytt to 0. /M) IJS/HCl buffer solution (p
lI 7. After sufficient equilibration with sO), centrifuge 1
Remove the N solution and add albuminosis solution 1 of various concentrations as mentioned above.
00d and shaken and stirred at IQ°C for 72 hours.

After shaking, the amount of absorbed M of albumin was determined by measuring the UV absorbance of the supernatant at 30 nm.

The results are summarized in Figure 1 in the form of adsorption isothermal storage.

Next, the entire M-absorbing phase body was separated into 0./M + J.
Hydrochloric acid buffer solution (PH7, sO) tθ0-! Wash thoroughly with t. Next, after centrifugation and removal of the adsorbed solution, /, j
0.1M Tris-HCl buffer solution (pH
7,jO)/00rttl, and the mixture was shaken and stirred at 70°C for 1 hour.

The amount of molten aluminum was determined by measuring the UV absorbance of the supernatant at z ro nm.

As a result, the elution rate was 75% or more in all cases.

Here, the elution rate (%)=(elution f/indicator)×100.

Application Example 2 The dye-supporting phase IA, 0rxt obtained in Example 7 was packed into a glass column with an inner diameter of A wx f3, and sufficiently equilibrated with a 2°C (mM) lithium-hydrochloric acid buffer solution (pH 7, jo).

Next itech serum albumin (Sigma reagent) r, z
q% α-lactalbumin (Sigma reagent) tt,
A 2-tynM) IJ-hydrochloric acid buffer solution (pH 7, to) containing S■ was added to the above column j.
After that, it was washed with 2jrr+M)l)nu-hydrochloric acid buffer solution (pH 7, jO) 2rd. Next'A Te2
J' ml of 0,2 j M NaC! Contains 1?
r mM) Lis-Saltㅅ□ buffer solution (pH 7,60), and further 2ψM / , j 1/I NaC1-containing 2j
eluted with a Lis-HCl buffer solution (pH 7, jO) (mM). All operations were performed at room temperature, and the flow rate was 8v10. The effluent was collected by a fraction collector, and each fraction was analyzed using liquid chromatography to calculate the concentration of each component. The results are shown in Figure-C.

In addition, the protein recovery rate is similar to that of human serum albumin compared to α-lactalbumin! It was r%.

From Figure 2, it can be seen that two proteins that are difficult to separate using an ion exchanger because their isogo points are close to each other can be clearly separated using the finger body of the present invention.

Application f! Bovine serum albumin was added to the concentration of J-J O1λW/V%.
, t M ) in Lis-HCl buffer solution (pH 7, rO).

Example~! The dye-supporting carrier obtained with! , θat is 0.1
After sufficient equilibration with M Tris/hydrochloric acid buffer solution (pH 7, SO), the mixture was centrifuged, the stabbing bath solution was removed, and the mixture was added to the albumin solution Tooy described above and stirred with shaking at 10° C. for 72 hours. After shaking, the supernatant was exposed to UV at 210 nm.
The adsorption amount of albumin was determined by measuring the absorbance, and it was found to be z, s (y-alfvan/ as one carrier).

Next, the adsorption carrier was completely crushed and separated, and 0.1 M)
It was thoroughly washed with a hydrochloric acid buffer solution (pH 7, jO) lOOIIj. Next, after centrifugation and removing the Uiflj liquid.

ノ, j M NaCl-containing Tris/HCl buffer solution (pH
7j0) It was added to 10θd, and the mixture was shaken and stirred at θ°C for several hours. The elution rate of albumin was determined to be 1% by measuring the UV absorbance at 2 r Onrh before the supernatant.

Application Example-μ A glass column (jacket +1) with an inner diameter of 1 oraad was filled with Ge θ-, a carrier obtained from a real rod Fl-/ having a particle size of 74t-201μ (average diameter/4t1μ). The height of the finger body is jutyl I and nine. Keep the column at 2J℃ with circulating water from the top of the column.

A 0.01M% pH 7.0 phosphorus buffer solution was flowed at a constant flow rate. When the packed bed becomes stable and the pointer of the pressure gauge attached to the top of the column becomes constant, read the self-loading value of the pressure gauge, and from that value, perform the above-mentioned operation in a so-called aerodynamic ram state without being packed with carrier. The pressure loss (Δp,
It was determined in the unit H7cd7socm-bea)'.

Dit speed (LV: i flow rate, unit "/f(r)t type k
When the pressure drop (Δp) was measured using KF, the results were shown in Figure 3. Within the measured flow velocity range (7 m/Hr or less as Lv), the Δp when using the carrier of the present invention was
A linear relationship is established between T and V, and LV = 7
Even when the liquid is passed at a high flow rate of m/Hr, the deformation of the object particles,
There was no evidence of crushing at all.

Instead of using the 45 body obtained in Comparative Example - Example -
3 Perform exactly the same operations as in Application Example-G except for using a phase in which G-A has been introduced, and calculate Δp and 1. The relationship between V was determined. The results are shown in Figure 3.

When using this cross-linked acarose-based carrier.

When the flow velocity exceeds Lv=1m/Rr, Δp starts to rise rapidly, and LV=2. Above s m/ar,
It became difficult to pass fluid through.

〔Effect of the invention〕

The carrier for affinity chromatography according to the present invention contains a glycerin monoester structure therein and is therefore highly hydrophilic.

Further, the substrate itself is characterized in that it is porous and entirely composed of aliphatic components, including the crosslinking agent, and contains no aromatic components. Therefore, it is a carrier in which a blue dye, which is an affinity ligand, is introduced into a highly hydrophilic porous substrate.

Mild elution conditions exhibit the effect of high protein recovery. Furthermore, the carrier according to the present invention has high mechanical strength and has little pressure loss when packed into a column.

Therefore, when the carrier according to the present invention is used in a column method,
Even if the height is increased, high flow rate processing is possible, making it an industrially useful carrier for affinity chromatography.

l Brief explanation of the screen Figure 1 shows the example-! FIG. 3 is a diagram showing the relationship between the adsorption amount of the dye-supporting carrier obtained in −≠ to bovine serum albumin and the equilibrium concentration.

FIG. 2 is a diagram showing the elution behavior when α-lactalbumin and bovine serum alpubano are separated using the dye-supported carrier obtained in Example-I″″C.

FIG. 3 shows the dye-bearing phase obtained in Example 7 and a cross-linked agarose-based carrier as a comparative example. FIG. 3 is a diagram showing the results of measuring the E power loss and the flow rate r, 1.

Sender: Mitsubishi Chemical Industries, Ltd. Representative patent attorney for long diameter - (7 others)

Claims (1)

[Claims] (1) The pore volume of the copolymer obtained from the crosslinking agent component selected from polyol polyacrylate and polyol polymethacrylate and the monomer component selected from glycidyl acrylate and glycidyl methacrylate is 0.2 in a dry state. After copolymerizing under conditions within the range of ~2.0 ml/g, (i) after hydrolyzing the epoxy groups in the obtained porous crosslinked copolymer, a dye represented by the following general formula (I) is obtained. or (ii) react the obtained porous crosslinked copolymer with the following general formula (
Method for producing a carrier for affinity chromatography, which comprises reacting with the dye represented by I) and then hydrolyzing the remaining epoxy groups in the porous crosslinked copolymer ▲ Numerical formulas, chemical formulas, tables, etc. There is▼・・・・・・( I
) (In the formula, one of R^1 and R^2 represents a hydrogen atom and the other represents a -SO_3^■ group)