JPH06134206A - How to separate biological material - Google Patents

Abstract

PURPOSE:To provide the method for separating a biological substance of high load under the condition of high flow velocity and low salt concentration by using an ion exchanger provided with a composite body structure carrying hydrophilic crosslinking gels in the pores of a porous body. CONSTITUTION:A vinyl polymer is highly preferable as a porous body, and as hydrophilic crosslinking gels carried on the pores of the porous body, crosslinking gels of polysaccharides treated with a crosslinking agent such as dextrin, agarose, chitosan or a hydrophilic synthetic polymer macromolecules such as polyvinyl alcohol or vinyl acetate are used. As a crosslinking agent, epichlorohydrin or glutaraldehyde or the like is used. A biological substance of 50g or more is loaded to 1 (one) litter of a separating agent comprising an ion exchanger such as dimethylaminoethyl. Then, elute of 0.02M or more to 0.2M or less is brought into contact with to prepare a desired biological substance. In the actual stage, the biological substance such as serum albumin, immunoglobulin, enzyme in an organism, DNA or peptide provided with physiological activity is separated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating biological substances such as peptides, proteins, nucleic acids and enzymes by liquid chromatography.

[0002]

2. Description of the Related Art When a biological substance represented by a protein is separated and purified, an ion exchanger composed of a crosslinked gel of a polysaccharide or an ion exchanger composed of a porous body of a synthetic polymer is generally used. However, this polysaccharide cross-linked gel has weak mechanical strength and a large pressure loss when used in chromatography, and it is difficult to pass the liquid at a linear velocity of, for example, 4 m / h or more. Furthermore, when a high-viscosity solution is passed, the resin may be deformed and consolidated due to the resistance of the solution. For this reason, it is necessary to take measures such as reducing the viscosity of the solution containing the biological substance by pretreatment, reducing the load, and preventing the biological substance concentrated to a high concentration from passing through the column during elution. .

Further, in order to elute the adsorbed biological substance, an eluent having a salt concentration of 0.2 M or more is generally required, which has a drawback that the load in the subsequent desalting operation becomes large. On the other hand, in the case of an ion exchanger made of a synthetic polymer porous material, the mechanical strength is satisfactory, but the amount of biomaterial that can be adsorbed is small, and 50% per liter of the separating agent.
It is difficult to load more than g.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a method for separating a high-loading amount of a biological substance, which has been impossible in the prior art, under the conditions of a high flow rate and a low salt concentration. is there.

[0005]

[Means for Solving the Problems] The present inventors have found that the above problems can be solved by using a specific separating agent. That is, the present invention has a composite structure in which a hydrophilic cross-linked gel is supported in the pores of a porous body and has a separating agent having an ion exchange group, and 50 g or more per 1 liter of the separating agent. The gist of the method is to separate the target biological substance by contacting with an eluent having a salt concentration of 0.02 M or more and 0.2 M or less after loading the mixture of biological substances of.

The present invention will be described in detail below. The separating agent in which an ion-exchange group is introduced into a composite in which a cross-linked gel is supported in the pores of the porous material used in the present invention is disclosed in JP-A 1-2-2.
As described in Japanese Patent No. 54247, it can be obtained by forming a complex of a porous body and a crosslinked gel, and further introducing an ion exchange group into the gel portion.

At this time, the pore volume of the porous body is preferably 30% or more and 90% or less of the resin volume. 30% or more and 70% or more in order to have a stronger structure that can withstand the passage of liquid at a high flow rate.
The following are more preferable. The pore diameter is preferably 0.01 μm or more and less than 1 μm. As the porous body, an organic polymer porous body or an inorganic porous body such as porous silicic acid is used. In particular, it is preferable to have a hydrophilic surface in order to carry a hydrophilic crosslinked gel. In the case of a porous body having a hydrophobic surface, a sufficient amount of the crosslinked gel cannot be carried in the pores, so that the amount of adsorbed biological substance decreases, which is not preferable.
Further, a porous body having a highly hydrophobic surface is not preferable because it has a drawback that nonspecific adsorption is likely to occur due to hydrophobic interaction with the biological substance to be separated.

Examples of the inorganic substance used as the porous material include hydroxyapatite and titanium oxide. On the other hand, the organic polymer porous body used as the porous body is preferably a vinyl copolymer or a polysaccharide porous body. The vinyl copolymer is specifically a cross-linked copolymer of a monounsaturated monomer having one vinyl group or an isopropenyl group in the molecule and a polyunsaturated monomer having two or more of those groups. It is a polymer. Moreover, as the monounsaturated monomer and the polyunsaturated monomer used for forming the porous body of the hydrophilic crosslinked copolymer, a hydrophilic surface can be formed after the copolymerization. Those having a hydrophilic group such as a hydroxyl group and a carboxyl group or capable of forming these by hydrolysis etc. (eg glycidyl group)
Is preferred.

A method for producing a porous body forming a composite used in the present invention from such a monounsaturated monomer and a polyunsaturated monomer is described in, for example, JP-A-60-96605. As described above, it can be easily produced by using a porosifying agent or the like. In the case of a polysaccharide porous body, a porous body such as agarose, chitosan, or cellulose is preferable. Furthermore, a cross-linked porous body having enhanced mechanical strength by being subjected to a cross-linking treatment with a cross-linking agent such as epichlorohydrin, diglycidyl compound and divinyl sulfone is more preferable.

As the porous body satisfying the requirements of the porous body forming the composite used in the present invention, a commercially available porous body as an existing adsorbent or an ion exchange resin may be used. It is possible. Among the above various porous materials, vinyl copolymers are particularly preferable. Further, the hydrophilic cross-linked gel supported on the pore portion of the porous body, dextran, agarose, a cross-linked gel of polysaccharide treated with a cross-linking agent such as chitosan and polyvinyl alcohol,
A crosslinked gel that is a hydrophilic synthetic polymer such as vinyl acetate and has a hydrophilic group such as a hydroxyl group or a carboxyl group, and is treated with a crosslinker such as epihalohydrin such as epichlorohydrin or an aldehyde such as glutaraldehyde is preferable. Particularly preferred is a cross-linked gel of polysaccharide.

The weight fraction of the crosslinked gel in the separating agent is preferably 10 to 30% by weight based on the dried separating agent. As a method for supporting the hydrophilic cross-linked gel in the pores of the porous body, there is a method of impregnating the porous body with an aqueous solution of a polysaccharide or a hydrophilic polymer and then reacting it with a crosslinking agent.

As a method for introducing an ion exchange group into the complex, a generally known method is used. Specifically, the ion-exchange group introduction reaction is performed on the cross-linked gel supported on the pores of the porous body, or the ion exchange group is introduced on the hydrophilic polymer before being supported on the pores of the porous body. It is obtained by carrying out a reaction. For example, when the ion-exchange group is a diethylaminoethyl group and the ion-exchange group-introducing reaction is carried out on the cross-linked gel supported on the pores of the porous body, the complex in a water-wet state is immersed in an alkaline aqueous solution of a predetermined concentration, Then, it is obtained by reacting with an aqueous solution of diethylaminoethyl chloride hydrochloride at 40 to 90 ° C. for 0.5 to 12 hours. The ion-exchange group may be introduced after reacting with an alkyl halide in an alkaline atmosphere.

The amount of ion exchange groups in the separating agent in the present invention is preferably 0.01 meq / ml or more and 0.3 or more.
Meq / ml or less, more preferably 0.02 me
It is q / ml or more and 0.2 meq / ml or less. The ion exchange group may be any of a weakly acidic group, a strongly acidic group, a weakly basic group and a strongly basic group, but a basic ion exchange group such as a diethylaminoethyl group is particularly preferable.

The volume (swelling degree) of 1 g of dried separating agent in water is preferably 2 ml / g or more and 7 ml / g or less. The biological substance in the present invention is a substance such as peptide, protein, nucleic acid, enzyme and the like, which is a water-soluble substance. More specifically, it is a protein such as serum albumin or immunoglobulin, an enzyme present in the living body, a physiologically active substance such as a protein produced by biotechnology, or a biological substance such as DNA or a peptide having a physiological activity. The biological substance to be treated in the present invention is a mixture of one of these and a contaminant, or a mixture of at least one of these and a contaminant, and the load on the separating agent is 50 g or more in total of the biological substance. . Molecular weight is 2
It is about 1,000,000 or less, and more preferably about 500,000 or less.

The separation operation is performed under the following conditions. For the adsorption operation, a solution containing a biological substance is prepared in an appropriate container, and even in the batch adsorption method in which a separating agent is added and brought into contact with the biological substance, the biological substance solution is passed through a column filled with the separating agent. The column adsorption method can also be used. Subsequent separation operations can be carried out by filling the column with a separating agent having adsorbed a biological substance by the batch adsorption method or by eluting the separating agent adsorbed by the column adsorption method as it is by a column chromatography method.

The amount of the biological material loaded on the separating agent is usually limited because the separating material has a small adsorption capacity for the biological material or weak mechanical strength and cannot pass a solution containing a high concentration of the biological material. Generally, the amount is less than 50 g per liter of the separating agent. The method for separating a biological substance using a separating agent according to the present invention separates a target biological substance from a contaminant under a high load condition of 50 g or more of the biological substance per liter of the separating agent.

In the present invention, in order to elute and recover the biological substance adsorbed on the separating agent, the separating agent is packed in a column,
An aqueous solution in which salt is dissolved is passed. Examples of the salt solution include salts of alkali metals such as sodium chloride, potassium chloride and lithium chloride. When the concentration of the salt is high, the amount of the salt to be removed becomes large in the desalting operation from the biological substance performed after the elution operation, which is not economically preferable. The salt concentration in the elution operation is 0.02M or more, 0.2M
The following are preferred. More preferably, 0.05M or more,
Concentrations below 0.15M are preferred.

The flow rate for passing the aqueous solution in which the salt is dissolved is
If it is too slow, the amount of biological substance that can be separated and purified per unit time is small, which is not preferable in terms of productivity. However, if it is too fast, the separation accuracy will be reduced, which is not preferable. In the present invention, the flow velocity of the aqueous solution in which the salt is dissolved is 0.5 at the linear velocity.
m / h or more and 30 m / h or less, preferably 2 m / h or more and 20 m / h or less, particularly preferably 4 m / h or more, 15
m / h or less.

[0019]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example 1] A composite of a porous body and a hydrophilic cross-linked gel was prepared according to the method described in JP-A-1-254247. Spherical porous polymer composed of 2,3-dihydroxypropyl methacrylate and ethylene glycol dimethacrylate (particle diameter 120 μm, pore diameter 1000 Å, pore volume 1.75 ml / g, specific surface area 15.4 m ² / g)
As a result, a complex carrying a crosslinked gel of dextran was obtained. Then, in the presence of NaOH, it is reacted with diethylaminoethyl chloride hydrochloride at 50 ° C. for 5 hours to obtain an ion exchanger having a diethylaminoethyl group (DEAE group) as an ion exchange group (exchange capacity of 0.11 meq / ml-resi.
n, swelling degree in water 3.81 ml / g-resin) was obtained. This ion exchanger was packed in a column having a diameter of 8 cm × 10 cm, and 3% of a 1% solution of a 1/1 mixture of bovine hemoglobin and bovine serum albumin (BSA) was prepared under the following conditions.
Elution was carried out at 0 ml with a flow rate of 10.7 m / h. Conditions: 0 to 6 minutes 0.01 M-phosphate buffer (pH
6.5) After 6 minutes 0.01M-phosphate buffer (pH 6.5) NaCl = 0.1M As a result, BSA was eluted after bovine hemoglobin, and the two components were completely separated. The elution time is 0.3-4.
It was 5 minutes and 8 minutes, respectively, and the recovery rates were 95% bovine hemoglobin and 99% BSA, respectively.

[Comparative Example 1] In the same manner as in the example, an attempt was made to separate a bovine hemoglobin / BSA mixture using an ion exchanger composed of cross-linked agarose (DEAE Sepharose Fast Flow, manufactured by Pharmacia). as a result,
Bovine hemoglobin eluted, but BSA remained adsorbed and did not elute.

Example 2 The ion exchanger of Example 1 was packed in a column having a diameter of 0.8 cm × 10 cm, and a 1/1 mixture of myoglobin and ovalbumin was used as a 1% solution.
Elution was performed under the conditions of 0 ml or less. Conditions: Flow rate 5.4 m / h 0 to 28 minutes 0.01 M-Tris hydrochloric acid buffer solution (pH
7.0) After 28 minutes 0.01M-Tris-HCl buffer (pH
7.0) NaCl = 0.15M As a result, ovalbumin was eluted following myoglobin, and the two components could be completely separated. The elution time is 0.5 to 20 minutes and 29 minutes, and 9 minutes of ovalbumin.
5% could be recovered. A chromatograph is shown in FIG. 1 below.

Example 3 A column having a diameter of 0.8 cm × 10 cm was packed with the ion exchanger of Example 1 and 70 ml of BSA as a 1% solution was passed under the following conditions to elute. Conditions: Flow rate 5.4 m / h 0 to 16 minutes 0.01 M Tris-HCl buffer (pH
8.0) 16 minutes or later 0.01 M-Tris-HCl buffer (pH
8.0) NaCl = 0.14M As a result, BSA elutes after 16 minutes and elution time is 1
The recovery rate was 8.5% at 96%.

[Example 4] The ion exchanger of Example 1 was packed in a column having a diameter of 0.8 cm x 10 cm, and 70 ml of a 1% solution of each protein was passed under the following conditions to elute. As a result, each protein was successfully recovered. The following table shows the NaCl concentration, elution time, and recovery rate during the elution operation.
Shown in 1. Conditions: Flow rate 5.4 m / h 0 to 16 minutes 0.01 M Tris-HCl buffer (pH
8.0) 16 minutes or later 0.01 M-Tris hydrochloric acid buffer solution (pH 8.0) containing NaCl at a predetermined concentration

[0024]

[Table 1]

[Example 5] 5 m of the ion exchanger of Example 1
l was dispersed in a 0.4% solution of BSA (0.01 M-Tris-HCl buffer, pH 7.0), allowed to stand at a liquid temperature of 5 ° C. for 6 hours, then taken out and placed in a column having a diameter of 0.8 cm × 10 cm. After filling, a buffer solution containing NaCl = 0.1 M was passed through at a flow rate of 5.4 m / h. Adsorbed BSA585
mg was recovered.

[0026]

EFFECT OF THE INVENTION The method of separating a biological substance using an ion exchanger having a composite structure in which a hydrophilic cross-linked gel is supported in the pores of a porous body of the present invention has a high load, high speed,
In addition, it is a method for separating and purifying a biological substance at a low salt concentration. Compared with conventional separation methods using ion exchangers, the amount of biological material processed in a single purification under high load is large,
Because it elutes at a high speed, the time required for the operation is small, and because the elution operation is performed at a low salt concentration, the load on the subsequent desalting operation is small, and a method for separating biological substances that is superior in terms of productivity and economy is provided. It is provided.

[Brief description of drawings]

FIG. 1 is a chromatograph when a mixture of myoglobin (A) and ovalbumin (B) was separated and purified using the ion exchanger used in Example 1 (Example 2).

[Explanation of symbols]

 A peak showing elution of myoglobin B peak showing elution of ovalbumin

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 30/48 G 8310-2J 30/88 E 8310-2J

Claims (1)

[Claims] 1. A separator having a composite structure in which a hydrophilic cross-linked gel is supported in the pores of a porous body and having an ion exchange group, and 50 g per 1 liter of the separator.
After loading the mixture containing the above biological substances, the salt concentration was adjusted to 0.
A method for separating a target biological substance by contacting an eluent in the range of 02M or more and 0.2M or less.