JPH01174505A - Production of composite separating agent - Google Patents

Abstract

PURPOSE:To produce a separating agent suitable for separation and purification of proteins, antibiotic substances, etc., having high mechanical strength, by granulating polyvinyl alcohol particles containing an aqueous salt solution and a separating agent consisting of hydrophilic polymer followed by reaction of the resultant granules with a crosslinking agent. CONSTITUTION:A separating agent consisting of a hydrophilic polymer with a giant network structure in a swelling state is dispersed in a dispersion medium consisting of an organic solvent (e.g., halogenated hydrocarbon) together with polyvinyl alcohol (pref. 200-2,000 polymerization degree and >=95mol.% saponification degree) and an aqueous salt(s) (e.g., NaCl) solution to provide polyvinyl alcohol granules containing the above-mentioned separating agent and aqueous salt(s) solution. Thence, the resultant granules are naturally gelled followed by reaction with a crosslinking agent (e.g., glyoxal) thus obtaining the objective such composite separating agent having normally 20-1,000mu granular diameter as to contain 40-80wt.% of a polysaccharides based separating agent of 5-500mu particle size in cross-linked polyvinyl alcohol granules containing macropores.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separating agent, and more specifically, it relates to a method for producing a complex separation agent, and specifically, it relates to a method for producing a composite separating agent, and more specifically, it relates to a method for producing a complex separation agent, and more specifically, it relates to a method for producing a composite separation agent. The present invention relates to a method for producing a composite separating agent suitable for separating and purifying.

[Conventional technology]

Traditionally, separation agents for separating and purifying biological substances include:
Separating agents made from polysaccharides such as dextran and agarose are known. Since these separation agents are highly hydrophilic, they are characterized by low nonspecific adsorption, especially with proteins.

Furthermore, it has a swelling degree in water of 10-440 g/f-dry, a huge network structure, and excellent separation ability. It can be said that it is a separation agent particularly suitable for gelte permeation chromatography.

[Problem that the invention seeks to solve]

However, although these separation agents have excellent separation ability, they are soft and have weak mechanical strength, and on an industrial scale that requires high-rises and high-flow rates, it is difficult to pass the liquid through due to compaction. The problem is that there are many cases where this is possible.

The present invention provides a method for producing a novel composite separation agent that maintains the excellent separation ability of a polysaccharide separation agent having a large network structure and improves the mechanical strength, which is a problem with the above-mentioned separation agents. The purpose is to provide.

Therefore, in order to improve the mechanical strength of the above-mentioned separating agent, we investigated a composite separating agent, and found that it met this purpose by incorporating it into a polyvinyl alcohol base having a macropore structure with a degree of cross-linking of ~10 mol. The present invention was completed by discovering that a composite separating agent that can be obtained can be obtained.

[Means for solving problems]

That is, in the present invention, a swollen separating agent made of a hydrophilic polymer having a large network structure is dispersed and spheroidized in a dispersion bath made of an organic solvent together with an aqueous solution of polyvinyl alcohol and a salt, and the separating agent and the salt aqueous solution are dispersed into balls. The gist of the present invention is to provide a method for producing a composite separation agent, which is characterized in that polyvinyl alcohol particles containing the following are obtained, the polyvinyl alcohol-2 particles are spontaneously gelled, and then reacted with a crosslinking agent.

The composite separation agent of the present invention has a particle size of 2Q to 7000μ,
≠0-10% polysaccharide separating agent with a particle size of 5-zooμ in cross-linked polyvinyl alcohol particles having macropores
(volume ratio).

The composite separating agent contains the polysaccharide separating agent by dispersing the swollen polysaccharide separating agent in a dispersion bath with an aqueous polyvinyl alcohol solution and forming balls, and then,
It can be produced by making balls by utilizing the natural gelation phenomenon of an aqueous polyvinyl alcohol solution, making it insolubilized, and then carrying out a crosslinking reaction.

The macropores of the crosslinked polyvinyl alcohol base are preferably larger than the pores of the giant network structure of the included polysaccharide separation agent, allowing biopolymers such as proteins and enzymes to freely enter and exit.

These macropores can be formed by utilizing the natural gelation phenomenon of an aqueous polyvinyl alcohol solution.

The natural gelation phenomenon referred to here means that an aqueous polyvinyl alcohol solution becomes insolubilized without using a crosslinking agent.

It has long been known that when an aqueous polyvinyl alcohol solution is left to stand, its viscosity increases over time and eventually gels.

This phenomenon is thought to occur due to hydrogen bonds between polyvinyl alcohol molecules, and the higher the concentration of the aqueous polyvinyl alcohol solution or the lower the temperature at which it is left, the faster gelation occurs. The reason why macropores are formed by manufacturing using natural gelation phenomenon is not clear, but it is probably due to polyvinyl alcohol precipitating from the reaction medium and phase separation due to natural gelation phenomenon, resulting in a heterogeneous structure. It is thought that this makes the material more porous.

The present invention relates to a method for producing a composite separating agent in which a polysaccharide-based separating agent is incorporated into a polyvinyl alcohol base having a macropore structure and a degree of crosslinking of from 10 to 10 mol. The method involves dispersing the swollen polysaccharide separating agent in a dispersion bath consisting of an organic solvent together with an aqueous solution of polyvinyl alcohol and salts to form balls.
The method is characterized in that polyvinyl alcohol particles containing the separating agent and a salt aqueous solution are obtained, and then the polyvinyl alcohol particles are spontaneously gelled and then reacted with a crosslinking agent. Examples of separating agents that can be used include crosslinked dextran having a large network structure, agarose, polysaccharide systems such as crosslinked agarose, and polyacrylamide systems obtained by copolymerizing acrylamide with N,N methylenebisacrylamide.

Since the separating agent is in the form of a dry powder, it is swollen with water and added to the aqueous solution of polyvinyl alcohol and salts.

Alternatively, it can be added to an aqueous polyvinyl alcohol solution to which salts have been added to cause swelling.

In order to swell the separating agent with water, it is necessary to swell the separating agent with water in a boiling water bath for 3 to 72 hours at room temperature, although the degree of swelling varies depending on the degree of swelling of each separating agent.
It takes 7 hours.

The polysaccharide separating agent can be incorporated into the polyvinyl alcohol base by dispersing the separating agent in a swollen state together with an aqueous polyvinyl alcohol solution to which salts have been added in a dispersion bath consisting of an organic solvent to form balls. can.

Polyvinyl alcohol has a polymerization degree of several tens to several tens, preferably 200-2000, a saponification degree of Q mol or more,
Preferably! Something more than that is used. Polyvinyl alcohol is 2-30%, preferably! It is dissolved in water to form a natural gel to a concentration of ~lj%. However, since it takes a long time for natural gelation to occur with only an aqueous polyvinyl alcohol solution, a salt is added to the aqueous polyvinyl alcohol solution in order to promote natural gelation. As the salt, one having the effect of precipitating and coagulating polyvinyl alcohol, such as sodium chloride and sodium sulfate, may be used.

The amount used varies depending on the type of salt, etc., but it is usually added at a concentration of 3% or more, within a range where polyvinyl alcohol does not precipitate.

In particular, the amount of sodium chloride added varies depending on the degree of polymerization, degree of saponification, and concentration of polyvinyl alcohol. −
/ flathead (preferably 3 to 10%), and when using a 7% polyvinyl alcohol aqueous solution, it is added in an amount of /-77% (preferably 1/3%).

Furthermore, natural gelation can be further promoted by adding an alkali such as sodium hydroxide to the polyvinyl alcohol aqueous solution to which a salt has been added.

Natural gelation is carried out after dispersion in an organic solvent together with a salt-added polyvinyl alcohol aqueous solution and a swollen polysaccharide separating agent.

The organic solvent usually includes aromatic hydrocarbons such as toluene, benzene, chlorobenzene, dichlorobenzene and their halogen derivatives, n-hebutane, n-hexane,
Aliphatic hydrocarbons such as liquid paraffin, cyclohexane, dichloromethane, dichloroethane, aliphatic hydrocarbons and their halogen derivatives, etc. are used. These organic solvents are usually used alone, but two or more types may be used in combination.

The amount of organic solvent used is 2 (2) of the polyvinyl alcohol aqueous solution.
(capacity) times or more, preferably 2 to 6 (capacity) times. In addition, dispersion stabilizers such as Toshite, ethyl cellulose, cellulose acetate butyrate, and ethyl hydroxyethyl cellulose, as well as oil-soluble dispersion stabilizers such as gum arabic, sorbitan sesquioleate, sorbitan monooleate, and sorbitan monostearate, are included in the organic solvent. It is preferable to add an agent.

The amount is usually 0.0r-10%, preferably 0.0% to the organic solvent. / -1%.

Natural gelation occurs at 0-10℃ after dispersion granulation, preferably from 3 to 10℃.
It is carried out at 30° C. for 2 to 200 hours, preferably 5-ioo hours.

After spontaneous gelation, a crosslinking reaction is performed.

As a crosslinking agent, a dialdehyde compound such as glyoxal, glutaraldehyde, terephthalaldehyde; /
, 2, 3. ≠-Jepoxy compounds such as jepoxybutane; ethylene glycol diglycidyl ether, /, Hiro-
Glycidyl ether compounds such as butanediol diglycyl ether; epihalohydrin compounds such as epichlorohydrin and shrimp bromohydrin, which can form a crosslinking moiety having two or more carbon atoms between polyvinyl alcohols, are used. Preferably, dialdehyde compounds are used in which acetalization crosslinks are formed. The crosslinking agent is
It is usually used in an amount of 1 mol or more based on the total hydroxyl groups of polyvinyl alcohol.

The crosslinking reaction is carried out by adding the above-mentioned crosslinking agent after natural gelation. Further, the crosslinking reaction can also be carried out in water after filtration of the spherical particles after natural gelation to separate the organic solvent and washing with acetone and methanol.

The crosslinking reaction is carried out at 0° C. to 100° C., preferably 30° C. to t0° C., for 1 hour to 20 hours, preferably 2 hours to t hours, after spontaneous gelation.

Let's do it. During the crosslinking reaction, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as hydroxide, potassium hydroxide, etc. is used as a catalyst depending on the crosslinking agent.

When using a dialdehyde compound as a crosslinking agent, an acid is used as a catalyst. The amount of acid used is determined by the concentration in the aqueous layer /N
It is preferable to add the following amounts. As a crosslinking agent,
When using a 2I-rohydrin compound, an alkali is used as a catalyst. The amount of alkali used is when the concentration in the water layer is 2.
Add so that the amount is N or higher. - During the crosslinking reaction, including when the crosslinking reaction is carried out in water as described above, it is preferable to add a salt or an aqueous salt solution such as sodium sulfate or IJum chloride.

The amount of salt added varies depending on the type, etc., but it is usually added at a concentration of 6 salts or more, which is higher than the concentration at which the polyvinyl alcohol aqueous solution causes precipitation and coagulation. For example, for sodium chloride, 20% or more,
For sodium sulfate, 4% or more is added.

The crosslinked polyvinyl alcohol particles thus obtained are separated by filtration. Next, it is washed with a water-miscible organic solvent such as acetone or methanol, and then thoroughly washed with water.

In order to completely remove the organic solvent, it is preferable to heat the separated crosslinked polyvinyl alcohol particles in water and distill off the organic solvent by azeotropic distillation.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to the following examples.

Example I Into an OlSt three-way flask equipped with a stirrer and a reflux condenser, dichloroethane xjOml was added, and cellulose acetate was added. A dispersion bath was prepared by dissolving 0.21 °C of thyrate (manufactured by Eastman Kodak Company, trade name: CAB 311-20).

Separately, put sodium chloride and t1 water jOtd into a 200-meter Mitsuro flask equipped with a stirrer and a reflux condenser, dissolve it while stirring, and add powdered polyvinyl alcohol (manufactured by Nippon Gosei Kagaku Kogyo Co., Ltd., trademark Gohsenol NL) to this. -01)2,j? Add and disperse? After heating at r°C for 1 hour to dissolve, 6-jN sodium hydroxide was further added and stirred.

The cross-linked dextran-based separation agent (manufactured by Pharmacia, trade name Sephadex G j O (Medium)) is
Add ST to a potassium chloride aqueous solution (sodium chloride, water) in a 200 d Mitsulo flask equipped with a stirrer and a reflux condenser, disperse and heat at 2°C for 7 hours.
After heating for a period of time to swell, it was added to the above polyvinyl alcohol solution which had been cooled to room temperature and stirred. Thereafter, it was dispersed in a dispersion bath consisting of the above organic solvent. l at room temperature
The mixture was allowed to stand for several hours with stirring to allow natural gelation.

After natural gelation, further add λj% saline solution ioo, z,
After stirring for 3 hours, filter and add acetone,
After washing with methanol, wash with water and add a stirrer again! 00tgl
Transfer to a Mitsuro flask, add 4 cOt of sodium chloride and 200.1 g of water, and add 2j% glutaraldehyde aqueous solution l≠yttl while stirring! N hydrochloric acid 4cal
was added and heated at 6J'C for 2 hours without stirring to carry out a crosslinking reaction. After cooling to room temperature, it was filtered and thoroughly washed with water. Milky white spherical crosslinked polyvinyl alcohol particles with an average particle size of 700μ and containing about 76 crosslinked dextran-based separating agents with an average particle size of 220μ were obtained.

Cross-linked dextran-based separating agent used in Examples (manufactured by Pharmacia, trademark Sephadex G100 (Medium))
The same operation as in Example 1 was carried out except that the type and amount of addition were changed as shown in Table 1.

Cross-linked dextran-based separating agent with an average particle size of 210 μm
Milky-white spherical crosslinked polyvinyl alcohol particles having an average particle diameter of 700 μm and containing 100% polyvinyl alcohol particles were obtained.

Example 3 In a 200 d three-meter flask equipped with a stirrer and a reflux condenser, put 100 d of sodium chloride and 100 d of water, dissolve with stirring, and add powdered polyvinyl alcohol (Gohsenol NH-/r) to this. ! ? , cross-linked dextran-based separation agent (Sephadex Gtso (SF)
) 2.39 was added and dispersed, and the mixture was heated at t° C. for 3 hours to dissolve the polyvinyl alcohol and swell the crosslinked dextran-based separation agent. Then, tN sodium hydroxide was further added and stirred.

After cooling to room temperature, the mixture was dispersed in the same organic solvent as in Example 1 except that the amount of cellulose acetate butyrate added was changed to 7.22 rt, and left to stand at room temperature with stirring for 1 hour to allow spontaneous gelation. I did it.

After natural gelation, add 20% sodium chloride aqueous solution 10
After adding 0- and stirring for 3 hours, it was filtered and 1. After washing with acetone and methanol, wash with water and transfer again to a 300rd Mitsuro flask equipped with a stirrer, add tog of water and stir while cooling to form granular hydroxide) After dissolving IJum 72f, add epichlorohydrin 3rd and stir. While heating at 60° C. for 6 hours, a crosslinking reaction was carried out. After cooling to room temperature, it was filtered and thoroughly washed with water, and then crosslinking reaction was performed once again under the same conditions. Average particle size 6
? Pale yellow spherical crosslinked polyvinyl alcohol particles with an average particle diameter of JjOμ containing about 70 μ crosslinked dextran-based separating agents were obtained.

Example ≠ Type of polyvinyl alcohol used, amount added, and! N
The same operation as in Example 3 was carried out except that the amount of sodium hydroxide added was changed as shown in Table 1. The average particle size is 3, which contains about 70 crosslinked dextran-based separating agents with an average particle size of 6rμ! Pale yellow spherical crosslinked polyvinyl alcohol particles of Oμ were obtained.

[Pressure loss measurement test]

A glass column (with a jacket) having an inner diameter of L21111φ was filled with the separating agent obtained in Example V, which had been sized to have a particle size of /≠−λ7μ. The layer height of the separating agent was jQcIIL. The column was maintained at 2J°C with circulating water, and distilled water was flowed from the top of the column at a constant flow rate. When the packed bed became stable and the pointer of the pressure gauge attached to the top of the column became constant, the scale of the pressure gauge was read at 9, and from that value, it was assumed that no separation agent was packed in the so-called aerodynamic ram state, as described above. The pressure loss (Δp unit Af/cil/-t O
an-bed) was determined.

When the pressure loss (Δp) was measured while varying the flow velocity (LV: linear flow velocity, unit: m/Hr), the results shown in FIG. 1 were obtained. Within the measured flow velocity range (3 m/H as LV)
When the separation agent of the present invention is used, a linear relationship is established between Δp and LV, and even when the liquid is passed at a high flow rate of LV=sm/Hr, no deformation or crushing of the carrier particles is observed. I couldn't.

For comparison, instead of using the separating agent obtained in Example≠, the included polysaccharide-based separating agent Sephadex c
The same operation as above was performed except that t, zo (M) (manufactured by Pharmacia) was used to determine the relationship between Δp and LV. The results are shown in Figure 1.

When using a polysaccharide-based separation agent, the flow rate is LV = 0.3
When Δp exceeded m/Hr, Δp began to rise rapidly, and at LV = 0.4 m/Hr or more, it became difficult to pass the liquid.

Application example / Example / Composite separation agent obtained with λ 10 simple φ x jO
OIII! After filling an IH glass column and thoroughly rinsing with water, the following operations were performed to create a calibration curve, and the performance as a support for Gelte chromatography was evaluated.

Aqueous solution of dextran with known molecular weight (concentration jW/V96
), 300 μL of an aqueous solution of polyethylene glycol (concentration JW/V%) was charged to the above column, and then distilled water was flowed at a flow rate of O≠ml/head to elute dextran and polyethylene glycol.

Dextran and polyethylene glycol in the effluent were detected using a differential refractometer, and their elution positions (peak top positions) were determined.

Figure 2 shows the results.

Application example The composite separating agent obtained in Example 3 and ψ was
Packed into a glass column of 1φXj00tmH, t j
OmM NaC! Containing SO mM phosphate buffer (
After sufficient equilibration at pH 7, ≠ O), the following operations were performed to create a protein calibration curve, and the performance as a support for gel gel chromatography was evaluated.

Proteins with different molecular weights shown below were dissolved in 10mM phosphate buffer (pH 7, ψO) containing /j OmM NaCL to a concentration of 0.1%, /J-0μt.
1 was charged to the above column. After charging, flow rate o,
At 2ytl/rHR/j Contains OmM NaCtj
The protein was eluted by flowing OmM phosphate buffer.

Protein detection was performed using a UV meter.

(J r Onm ) For comparison, the included polysaccharide separation agent Sephadex Gl! Perform the same operation as above using 0(M),
A protein calibration curve was determined.

Kav was determined from the elution position of the protein using the following formula, and this value was plotted against the molecular weight, and is shown in FIG. 3 as a calibration curve.

(Note that Vo is the value determined using blue dextran with a molecular weight of approximately 2 million.) From Figure 2, the calibration curve obtained using the separation agent of the present invention shows good linearity, and is suitable for gel filtration chromatography. It can be seen that it is suitable as a carrier.

Figure Nakaya Protein Molecular Weight (×1O3) Isoelectric Point/: Thyroglobulin 667 ≠, j2: Ferritin Hiro170 4'~j3:
Bovine serum r-globulin l! Ta 1, 2-7
．． 3V: Bovine serum Fulbumi/64. J
4(j ~4(, rj = ovalbumin
≠j 4C046: Myoglobin
i7. t t, t to t, λ″7 [Effect] The composite separating agent obtained by the method of the present invention is useful as a packing material for chromatography, particularly for aqueous gel permeate chromatography.

The composite separated material obtained by the method of the present invention has a particle size of 20 to
The polysaccharide separation agent with a particle size of j to 200μ is contained in crosslinked polyvinyl alcohol particles with a particle size of 1000μ and macropores at a ratio of 0 to 10% (volume ratio). Because of its high properties, it is characterized by low nonspecific adsorption, especially with proteins. Therefore,
For example, it is useful as a separating agent for various proteins such as r-' globulin, albumin, and myoglobin.

In addition, the composite separating agent of the present invention has excellent mechanical strength due to the crosslinked structure of the polyvinyl alcohol base.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of measuring the relationship between pressure drop and flow rate using the composite separation agent obtained in Example≠ of the present invention and a polysaccharide separation agent as a comparative example. In the figure, the horizontal axis is the liquid flow rate (-/hr, ), and the vertical axis is the pressure loss (kf
/cr/l −j Ocm bed ). Figure 2 shows the calibration curve of dextodin and polyethylene glycol for the composite separating agent obtained in Examples 1 and 2 of the present invention, and Figure 3 shows the composite separating agent obtained in Example 3 and ≠ and a comparative example. This is a protein calibration curve when a polysaccharide-based separation agent is used as the protein. In FIGS. 2 and 3, the horizontal axis represents the retention capacity, Kav, value expressed by the following formula, and the vertical axis represents the molecular weight. (Vo is the value determined using blue dextran with a molecular weight of approximately -2 million.) Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney - (1 other person) Figure 1: M, V, ( m/Ar) Fig. 2 Mamochi e-(Ryo Fig. 3 av

Claims (4)

[Claims] (1) A swollen separating agent made of a hydrophilic polymer having a giant network structure is dispersed and spheroidized in a dispersion bath made of an organic solvent together with an aqueous solution of polyvinyl alcohol and a salt to contain the separating agent and the salt aqueous solution. 1. A method for producing a composite separation agent, which comprises obtaining polyvinyl alcohol particles, which are then naturally gelled, and then reacted with a crosslinking agent. (2) The manufacturing method according to claim 1, wherein the crosslinking agent is a dialdehyde compound. (3) The manufacturing method according to claim 1 or 2, wherein the crosslinking reaction is carried out in the presence of an acid catalyst. (4) The manufacturing method according to claim 1, 2, or 3, wherein the dispersion bath is a halogenated hydrocarbon.