JPH0722701B2 - Biochemical separation agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a pyrogen, a nucleic acid, and a nucleic acid-related substance which are improved so as to separate biochemical substances selected from pyrogens, nucleic acids, and nucleic acid-related substances with high efficiency. It relates to a biochemical separation agent of choice.

[Conventional technology]

Biochemical substances such as pyrogens, nucleic acids, and nucleic acid-related substances may need to be separated from raw materials as useful substances, or may need to be removed as impurities from pharmaceuticals. Technology for separating these biochemical substances Is considered an important technology in related fields.

As a conventional method for separating these biochemical substances, for example, as disclosed in JP-A-57-171403, a method of separating using a separation membrane, and a method disclosed in JP-A-54-67024 are disclosed. A method of adding hyaluronate to the above and treating with an anion resin, a method of enzymatically degrading biochemical substances present as impurities as disclosed in JP-A-47-20352, and JP-A-46-529. A method of gelling using a gel made by polymerization of a derivative obtained by the reaction of a biochemical substance which separates with a compound which reacts biochemically as disclosed in JP-A-59-39297. As disclosed, a method of reacting with a basic water-soluble polymer to cause precipitation, a method of using an ion exchange resin as disclosed in JP-A-52-99291, or the presence of an acid or an alkali as impurities To separate biochemicals that Law, and the like. However, in the conventional method, pyrogen, nucleic acid,
It has been difficult to effectively remove biochemical substances selected from the nucleic acid-related substances.

In these methods, there are cases where it is difficult to sufficiently remove impurity biochemical substances with only one operation, and cases where the separation from useful drugs is insufficient.

Further, when a biochemical substance existing as an impurity is enzymatically or chemically decomposed, there is a drawback that other drugs themselves are also decomposed.

In order to improve the drawbacks of these methods, for example, when extracting useful proteins or amino acids from the cell extract, protamine sulfate obtained from salmon as a separating agent for removing coexisting nucleic acids, or obtained from crab. It is known that chitosan is used (Biochemistry Experiment Course 5, P201, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin). The mechanism of nucleic acid removal in this case is as follows: When an aqueous solution containing nucleic acid is mixed with an aqueous solution in which protamine sulfate as a nucleic acid remover or chitosan is dissolved, the negative ion of the phosphate group of the nucleic acid and the positive ion of the nucleic acid remover are By binding to form polymeric aggregates. By removing this aggregate, an aqueous solution from which nucleic acid has been removed can be obtained.

However, since protamine sulfate and water-soluble polymers such as chitosan are water-soluble, compared with biochemical substances such as nucleic acids contained in the solution, which need to be removed, a water-soluble separating agent is used. When used in excess, some separating agent remains in the solution after removing the aggregates.

There were cases where these biochemical separation agents were used for applications where there is no problem even if these water-soluble biochemical separation agents remain. Since there are many applications where residual substances are not allowed, the target biochemical substance can be separated with sufficient efficiency in just one operation.
Moreover, the appearance of a biochemical separating agent that does not leave the biochemical separating agent in the solution after separation is desired.

[Problems to be solved by the invention]

The present invention has been made in view of the above circumstances, and a biochemical substance selected from a pyrogen, a nucleic acid, and a nucleic acid-related substance can be separated with sufficient efficiency by only one operation, and coexist with other substances. Improved biochemical separation agent selected from pyrogens, nucleic acids, and nucleic acid-related substances so that the chemical agent does not undergo decomposition and the biochemical separation agent does not remain in the solution after separation. The purpose is to provide.

[Means for solving problems]

That is, the present invention provides (1) a separating agent for a biochemical substance obtained from a pyrogen, a nucleic acid, and a nucleic acid-related substance, which comprises a crosslinked quaternary nitrogen-containing water-soluble polymer, and (2) the above-mentioned crosslinked substance. The quaternary nitrogen-containing water-soluble polymer is a crosslinked cationic hydroxyalkyl cellulose, a separating agent for a biochemical substance selected from a pyrogen, a nucleic acid, and a nucleic acid-related substance. 4th
The water-soluble polymer containing quaternary nitrogen is a cross-linked cationic hydroxyethyl cellulose, a separating agent for biochemical substances selected from pyrogens, nucleic acids, and nucleic acid-related substances, (4) cross-linked quaternary nitrogen There is provided a biochemical separating agent selected from a pyrogen, a nucleic acid, and a nucleic acid-related substance, wherein the contained water-soluble polymer is a crosslinked cationic chitosan.

The biochemical substance to be separated in the present invention is selected from pyrogens, nucleic acids, and nucleic acid-related substances.

In the present invention, what is called a pyrogen is a general term for substances that generate fever when administered to a living body, and is also called a pyrogen or a pyrogen. There are two types of pyrogens, extrinsic and endogenous. Exogenous pyrogens can be broadly classified into exotoxins (exotoxins) secreted by pathogenic bacteria such as diphtheria and Staphylococcus aureus, and endotoxins, which are components of the cell wall of Gram-negative bacteria such as Escherichia coli. The endotoxins of Gram-negative bacteria are
Lipopolysaccharide, a complex of lipids bound to polysaccharides (LP
S). For endogenous pyrogens,
In addition to lymphokines and macrokines such as interleukin 1 and interferon, there are chemical mediators and prostaglandins secreted by neutrophils, eosinophils, macrophages, mast cells and the like. The pyrogens to be separated in the present invention are not limited to the above examples.

What is called a nucleic acid in the present invention is a polynucleotide consisting of phosphate, pentose, and base. It contains either ribose or deoxyribose as the pentose, and the former is called ribonucleic acid (RNA) and the latter is called deoxyribonucleic acid (DNA). Nucleic acid is structurally and functionally D
There are two major categories, NA and RNA. All are characterized by the composition of four major bases, adenine, guanine and cytosine are common base components in DNA and RNA, and in DNA, thymine and RNA are other components.
Contains uracil. In higher cells, DNA is localized in the chromosome of the cell nucleus and becomes the gene body, and RNA is found distributed throughout the cell, and it is a worker of protein biosynthesis, which is the gene expression of the gene.

In the present invention, what is called a nucleic acid-related substance is a nucleoside, a nucleotide, or a derivative thereof which is a component of nucleic acid, and occupies an important position as a seasoning or a medicine. These products are industrially produced by fermentation or synthetic methods. For example, 5'-inosinic acid and 5'-guanylic acid are used as seasonings, and inosine, ATP, CDP-choline, FAD, N
AD, coenzyme A, etc. are widely used as medicines.
Some nucleic acid derivatives affect DNA synthesis in cells, and those having a mutagenic effect particularly need to be removed as impurities from the viewpoint of safety.

The separating agent for biochemical substances selected from the pyrogens, nucleic acids, and nucleic acid-related substances of the present invention means the above-mentioned pyrogens,
Separation agents used for the purpose of separating these biochemical substances from a mixture containing one or more biochemical substances selected from nucleic acids and nucleic acid-related substances (hereinafter simply referred to as biochemical substance separation agents) There is also).

In the present invention, the quaternary nitrogen-containing water-soluble polymer means that the molecule of the water-soluble polymer has a moiety in which four hydrocarbon residues and one acid group are bonded to the nitrogen atom. It is a water-soluble polymer having a different structure and is called a quaternary ammonium salt. The nitrogen atom of the ammonium salt is a so-called pentavalent nitrogen, but it is bound to four hydrocarbon residues by a covalent bond to form a monovalent cation, which is bound to an acid group by an ionic bond. Is. The crosslinked quaternary nitrogen-containing water-soluble polymer is a product obtained by crosslinking the above quaternary nitrogen-containing water-soluble polymer.

Among the cross-linked quaternary nitrogen-containing water-soluble polymers, the most preferable one is cross-linked cationic raw hydroxyalkyl cellulose. When the crosslinked cationic hydroxyalkyl cellulose is used as a biochemical separation agent, a higher separation effect is obtained than when other crosslinked quaternary nitrogen-containing water-soluble polymer is used. The present invention is described in detail using the example of a cross-linked cationized hydroxyalkyl cellulose.

The method for producing the crosslinked cationic hydroxyalkyl cellulose used in the present invention is not particularly limited, and for example, the cationized hydroxyalkyl cellulose disclosed in Japanese Patent Application No. 61-217672 (Japanese Patent Application Laid-Open No. 63-72701) can be prepared. In the production method, it is advantageous in terms of reactivity and economy to add a cross-linking agent and react when adding a cationizing agent.

Further, as disclosed in JP-A-56-62801, cellulose ether is reacted with glycidyltrialkylammonium halide in a mixed solvent consisting of water and a soluble organic solvent to crosslink the resulting cationic cellulose ether. You may cross-link with an agent and use it.

Examples of the alkylene oxide used for producing the crosslinked cationic hydroxyalkyl cellulose include ethylene oxide, propylene oxide and various butene oxides.

By changing the addition amount of this alkylene oxide, hydroxyalkyl cellulose having various degrees of substitution can be obtained.

Depending on the type of alkylene oxide used, a crosslinked product of cationic hydroxyethyl cellulose, cationic hydroxypropyl cellulose, and cationic hydroxybutyl cellulose can be obtained. Of these, crosslinked cationic hydroxyethyl cellulose is most preferable, it is easy to handle, and the highest separation effect is obtained. As the cationizing agent used for producing the crosslinked cationic hydroxyalkyl cellulose, glycidyltrialkylammonium halide or its halohydrin type can be used. For example, glycidyl trimethyl ammonium chloride, glycidyl triethyl ammonium chloride, glycidyl trimethyl ammonium bromide, 3-chloro-2-hydroxypropyl trimethyl ammonium chloride and the like.

Examples of the crosslinking agent for cationized hydroxyalkyl cellulose include epichlorohydrin, 1,3-dichloro-2-propanol, 1,4-bis- (2,3, epoxypropoxy) -butane.

Further, the amount of the cross-linking agent used is generally desired to be about 1% to 20% of the number of moles of glucose. If the amount of the cross-linking agent used is outside this range, the separation performance is not sufficient.

A method of using a separating agent for a biochemical substance selected from pyrogens, nucleic acids, and nucleic acids will be described using an example of a method for separating nucleic acids using crosslinked cationic hydroxyalkyl cellulose.

The crosslinked cationic hydroxyalkyl cellulose obtained by the above-mentioned production method or the like is added to an aqueous solution containing a nucleic acid or a cell extract, and then thoroughly stirred using a stirrer or the like. Then, the mixture is allowed to stand to precipitate the crosslinked cationic hydroxyalkyl cellulose adsorbing the nucleic acid to obtain a supernatant thereof. Alternatively, the crosslinked cationic hydroxyalkyl cellulose is separated by over-centrifugation,
Nucleic acids can be separated by a method of obtaining the supernatant. The addition amount of the cross-linked cationized hydroxyalkyl cellulose is adjusted depending on the nucleic acid content of the nucleic acid-containing solution to be removed, but generally, the addition amount of the cationic group is equal to or more than the number of moles of the nucleic acid. desirable.

The separating agent for biochemical substances of the present invention can be used, for example, in the use method as described in the above examples, but is not limited to the above. As another example of the method of use, for example, a method of packing a column with a crosslinked cationic hydroxyalkyl cellulose and using it may be used. In this case, when a solution containing nucleic acid is flown over the crosslinked cationic hydroxyalkyl cellulose packed in the column, the effluent becomes a solution containing no nucleic acid.

Although the case of separating nucleic acids as described above has been described in detail, the method of using these biochemical separating agents is as follows.
Even in the case of separating a nucleic acid-related substance or a pyrogen, exactly the same usage method can be applied.

〔The invention's effect〕

The biochemical separation agent of the present invention can separate biochemical substances selected from pyrogens, nucleic acids, and nucleic acid-related substances with sufficient efficiency by a single operation, as will be apparent from the following examples. It has excellent properties such that it can be formed, and that other coexisting agents are not decomposed, and that the separating agent for the biochemical substance does not remain in the solution after separation.

The reason why the biochemical separation agent does not remain in the solution after separation is that the crosslinked quaternary nitrogen-containing water-soluble polymer is sufficiently crosslinked, and may not swell in the aqueous solution. However, it never elutes in the aqueous solution and remains in the aqueous solution.

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Comparative Example 1) A 2% solution was prepared using a commercially available nucleic acid remover (Catalog No. C-7, Kurita Kogyo) using chitosan.

1 cc of this aqueous solution was added to 1 mg of calf thoracic cord nucleic acid in 0.15 M NaCl, 10 c
Add to the solution dissolved in c and stir with a stirrer for 10 minutes.
After that, the precipitate was centrifuged at 5000 rpm for 30 minutes to separate the supernatant. When the amount of nucleic acid contained in this supernatant was measured, 95% of the nucleic acid was removed.

However, when the content of the nucleic acid removing agent remaining in this supernatant was measured, it was found to be 30% of that of the original nucleic acid removing agent added to the nucleic acid solution.
% Remained in the supernatant.

Comparative Example 2 A 2% aqueous solution of cationized hydroxyethyl cellulose (degree of cationizing agent substitution: 1.5) using glycidyltrimethylammonium chloride as a cationizing agent was prepared. 1 cc of this aqueous solution was added to 1 mg of calf thorax nucleic acid to 0.15 M NaC.
Add to the solution dissolved in l, 10cc and stir with a stirrer for 10 minutes. Centrifuge the precipitate for 30 minutes at 5000 rpm,
The supernatant was collected. When the amount of nucleic acid contained in this supernatant was measured, 98% of the nucleic acid was removed.

However, when the content of cationized hydroxyethyl cellulose remaining in the supernatant was measured, 20% of the original nucleic acid removing agent added to the nucleic acid solution remained in the supernatant.

Example 1 Cationized hydroxyethyl cellulose using glycidyltrimethylammonium chloride as a cationizing agent (the degree of substitution of the cationizing agent is 1.1 and the degree of substitution of the hydroxyethyl group is 1.7) is 1,3-dichloro-2- which is a crosslinking agent. Propanol was added to 2% of the number of moles of glucose to react and crosslink.

This crosslinked cationized hydroxyethyl cellulose.
1 g of calf thoracic cord nucleic acid 1 mg is added to a solution prepared by dissolving 0.15 M NaCl, 10 cc, and then stirred with a stirrer for 10 minutes. After that, the crosslinked gel was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected.

When the amount of nucleic acid contained in this supernatant was measured, 98% of the nucleic acid was removed.

Further, no crosslinked cationized hydroxyethyl cellulose was present in this supernatant.

Example 2 Cationized hydroxyethyl cellulose using glycidyltrimethylammonium chloride as a cationizing agent (the degree of substitution of the cationizing agent is 1.1 and the degree of substitution of the hydroxyethyl group is 1.7) is added to epichlorohydrin, which is a cross-linking agent, with respect to the number of moles of glucose. 2% was added to react and crosslink.

This crosslinked cationized hydroxyethyl cellulose.
1 g of calf thoracic cord nucleic acid 1 mg is added to a solution prepared by dissolving 0.15 M NaCl, 10 cc, and then stirred with a stirrer for 10 minutes. After that, the crosslinked gel was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected.

When the amount of nucleic acid contained in this supernatant was measured, 98% of the nucleic acid was removed.

Further, no crosslinked cationized hydroxyethyl cellulose was present in this supernatant.

Example 3 Cationized hydroxyethyl cellulose using glycidyltrimethylammonium chloride as a cationizing agent (the degree of substitution of the cationizing agent is 1.1 and the degree of substitution of the hydroxyethyl group is 1.7) is 1,3-dichloro-2- which is a cross-linking agent. Propanol was added to 2% of the number of moles of glucose to react and crosslink.

This crosslinked cationized hydroxyethyl cellulose.
1 g was packed in a plastic mini-column, washed with distilled water from the top, and 1 mg of calf thoracic cord nucleic acid was added to 0.15 MN.
A solution dissolved in aCl, 10 cc was flown and the effluent was collected. When the amount of nucleic acid contained in this effluent was measured, it was 99.5.
% Nucleic acid was removed.

Moreover, no crosslinked cationized hydroxyethyl cellulose was present in this effluent.

(Example 4) Cationized hydroxyethyl cellulose using 3-chloro-2-hydroxypropyltrimethylammonium bromide as a cationizing agent (substitution degree of cationizing agent: 0.
6, Substitution degree of hydroxyethyl group 1.7) 1,3 which is a cross-linking agent
-Dichloro-2-propanol was added in an amount of 2% relative to the number of moles of glucose to cause a reaction and crosslink.

This crosslinked cationized hydroxyethyl cellulose.
1 g of calf thoracic cord nucleic acid 1 mg is added to a solution prepared by dissolving 0.15 M NaCl, 10 cc, and then stirred with a stirrer for 10 minutes. After that, the crosslinked gel was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected.

When the amount of nucleic acid contained in this supernatant was measured, 96% of the nucleic acid was removed.

Further, no crosslinked cationized hydroxyethyl cellulose was present in this supernatant.

Claims (4)

[Claims] 1. A separating agent for a biochemical substance selected from a pyrogen, a nucleic acid, and a nucleic acid-related substance, which comprises a crosslinked quaternary nitrogen-containing water-soluble polymer. 2. The pyrogen, nucleic acid, and nucleic acid-related substance according to claim 1, wherein the crosslinked quaternary nitrogen-containing water-soluble polymer is a crosslinked cationic hydroxyalkyl cellulose. Separation agent for biochemical substances selected from 3. The pyrogen, the nucleic acid, and the nucleic acid-related substance according to claim 1, wherein the crosslinked quaternary nitrogen-containing water-soluble polymer is a crosslinked cationic hydroxyethyl cellulose. Biochemical separation agent of choice. 4. The crosslinked quaternary nitrogen-containing water-soluble polymer is a crosslinked cationic chitosan, which is selected from the pyrogen, the nucleic acid, and the nucleic acid-related substance according to claim 1. Separation agent for biochemical substances.