JPH0199646A - Separating agent for biochemical substance - Google Patents

Abstract

PURPOSE:To obtain the title separating agent capable of separating a biochemical substance selected from pyrogens, nucleic acids, and nucleic acid-related substances with high efficiency with only one operation by composing the agent out of a cross-linked quaternary nitrogen-contg. water-soluble polymer. CONSTITUTION:A water-soluble polymer of the structure having a part wherein four hydrocarbon residues and one acid radical are bonded to a nitrogen atom. is cross-linked in the molecules of a water-soluble polymer, and a cross-linked quaternary nitrogen-contg. water-soluble polymer is obtained. When the obtained polymer is used as the separating agent for a biochemical substance selected from pyrogens, nucleic acids, and nucleic acid-related substances, the biochemical substance can be efficiently separate with only one operation. Furthermore, the separating agent does not dissolve other coexistent chemicals, and is not left in the soln. after separation. Cationic hydroxyalkylcellulose cross-linked with 1,3-dichloro-2-propanol, etc., is most preferably used as the cross-linked quaternary nitrogen-contg. water-soluble polymer.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention has been improved to separate biochemical substances selected from pyrogienes, nucleic acids, and nucleic acid-related substances with high efficiency. The present invention relates to a separation agent for biochemical substances selected from the following.

[Conventional technology]

Biochemical substances such as nosoilonoene, nucleic acids, and nucleic acid-related substances may need to be separated from raw materials as useful substances or removed as impurities from pharmaceuticals, etc., and technology to separate these biochemical substances is required. is considered an important technology in related fields.

Conventional methods for separating these biochemical substances include, for example, a method of separating door to door using a separation membrane as disclosed in JP-A-57-171403, and JP-A-54-6702.
A method of adding hyaluronate and treating with an anion resin as disclosed in JP-A-47-2035
A method for enzymatically degrading a biochemical substance present as an impurity as disclosed in JP-A-46-529, and a compound that biochemically reacts with the biochemical substance to be separated as disclosed in JP-A-46-529. A method of gel filtration using a glue produced by polymerization with a derivative obtained by reaction with a derivative, a method of precipitating by reacting with a basic water-soluble polymer as disclosed in JP-A-59-39297, Japanese Unexamined Patent Publication No. 52-
There are methods such as using an ion exchange resin as disclosed in No. 99291, or adding acid or alkali to decompose biochemical substances present as impurities. However, with conventional methods, it has been difficult to effectively remove biochemical substances selected from gyronoene, nucleic acids, and nucleic acid-related substances.

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

Furthermore, when biochemical substances present as impurities are decomposed enzymatically or chemically, there is a drawback that other drugs themselves are also degraded.

In order to improve the shortcomings of these methods, for example, when extracting useful proteins and amino acids from cell extracts, protamine sulfate obtained from salmon or protamine sulfate obtained from crab can be used as a separation agent to remove coexisting nucleic acids. (Biochemistry Experiment Course 5. P2O
1, Japan Biochemical Wire Mesh, Tokyo Kagaku Doujin). The mechanism of nucleic acid removal in this case is that when an aqueous solution containing nucleic acids is mixed with an aqueous solution in which the nucleic acid removing agent protamine sulfate or chitosan is dissolved, the negative ions of the phosphate group of the nucleic acid and the positive ions of the nucleic acid removing agent are mixed. By binding and producing macromolecular aggregates. By removing this aggregate, an aqueous solution free of nucleic acids can be obtained.

However, since water-soluble polymers such as protamine sulfate and chitosan are water-soluble, they require less water-soluble separation agents than biochemical substances that must be removed, such as nucleic acids, contained in solutions. If an excess amount is used, some separation agent will remain in the solution after removing the aggregates.

These water-soluble biochemical substance separation agents were sometimes used in applications where there was no problem even if they remained; Since there are many applications in which residual substances cannot be tolerated, target biochemical substances can be separated with sufficient efficiency simply by a single operation.
Moreover, there is a desire for a biochemical substance separating agent that does not remain in the solution after separation.

[Problem that the invention seeks to solve]

The present invention has been made in view of the above circumstances, and it is possible to separate biochemical substances selected from pyrotene, nucleic acids, and nucleic acid-related substances with sufficient efficiency, and moreover, to separate biochemical substances selected from pyrotene, nucleic acids, and nucleic acid-related substances with sufficient efficiency. An improved separation agent for biochemical substances selected from pyrogienes, nucleic acids, and nucleic acid-related substances, which does not cause the drugs to be degraded and does not leave any biochemical separation agents in the solution after separation. The purpose is to provide

[Failure to solve the problem]

That is, the present invention provides (1) a separation agent for biochemical substances selected from gylodiene, nucleic acids, and nucleic acid-related substances, comprising a crosslinked quaternary nitrogen-containing water-soluble polymer; (2) a crosslinked quaternary nitrogen-containing water-soluble polymer; The quaternary nitrogen-containing water-soluble polymer is a cross-linked cationic hydroxyalkyl cellulose. - A separation agent for biochemical substances selected from gyronoene, nucleic acids, and nucleic acid-related substances; (3) the above-mentioned The cross-linked quaternary nitrogen-containing water-soluble polymer is cross-linked cationic hydroxy-7-ethyl cellulose. A separation agent for biochemical substances selected from yronoene, nucleic acids, and nucleic acid-related substances, (4) a pyrodiene, a nucleic acid, characterized in that the crosslinked quaternary nitrogen-containing water-soluble polymer is a crosslinked cationic chitosan; , and a separating agent for biochemical substances selected from nucleic acid-related substances.

The biochemical substances to be separated in the present invention are selected from nitrogen, irogens, nucleic acids, and nucleic acid-related substances.

In the present invention, ceylonoene is a general term for substances that cause fever when administered to a living body, and is also called a pyrogen or a pyrogenic factor. There are two types of pyrotsuene: exogenous and endogenous. Exogenous pyrogienes can be broadly divided into exotoxins (endotoxins) secreted by pathogenic bacteria such as Diphtheria bacterium and Staphylococcus aureus, and endotoxins (endotoxins) which are components of the cell walls of Durham-negative bacteria such as Escherichia coli. What constitutes the endotoxin of Durham-negative bacteria?
Lipopolysaccharide (LP) is a complex of lipids bound to polysaccharides.
S). Endogenous 2-ylogenes include interleukin-1, interferon, lymphocytes and macrokines, as well as chemical mediators and grosstagrans secreted by neutrophils, eosinophils, macrophages, and mast cells. There are gins, etc. The 4-ylonoene to be separated in the present invention is not limited to the above examples.

In the present invention, what is referred to as a nucleic acid is a polynucleotide consisting of a phosphoric acid, a pentose, and a base. It contains only either ribose or deoxylitase as an nucleotide; the former is called IJg nucleic acid (RNA), and the latter is called deoxylysonucleic acid (DNA).

Nucleic acids are structurally and functionally divided into two types: DNA and RNA. All are characterized by the composition of four main bases,
Adeni/, guanine and cytosine are DNA, RN
A basic component common to A, DNA also contains thymine, and RNA contains urafur. In higher cells, DNA is localized in the chromosomes of the cell nucleus and serves as the gene body, while RNA is found distributed throughout the cell and plays a role in protein biosynthesis, which is the expression of genes.

In the present invention, substances related to nucleic acids refer to nucleosides and nucleotides, which are constituent components of nucleic acids.

and its derivatives, which occupy an important position as seasonings and medicines. These substances are industrially produced by fermentation or synthesis methods, such as 5'-inosinic acid,
5'-guanylic acid as a seasoning, inosine, ATP
, CDP-choline, I'AD, NAD.

Coenzyme A and the like are widely used as medicines. Some nucleic acid derivatives affect cellular DNA synthesis, especially those that have mutagenic effects.
For safety reasons, there are some impurities that need to be removed.

The agent for separating biochemical substances selected from pyrothienes, nucleic acids, and nucleic acid-related substances of the present invention is a mixture containing one or more biochemical substances selected from the above-mentioned pylotenes, nucleic acids, and nucleic acid-related substances. It refers to a separating agent used to separate these biochemical substances (in the following text, it may simply be referred to as a separating agent for biochemical substances).

In the present invention, a quaternary nitrogen-containing water-soluble polymer is defined as a water-soluble polymer having a portion in which four hydrocarbon residues and one acid group are bonded to a nitrogen atom in the molecule. It is a water-soluble polymer with a similar structure, and is called a quaternary ammonium salt. The nitrogen atom of ammonium salt is so-called pentavalent nitrogen, but it bonds with four hydrocarbon residues through covalent bonds to form a monovalent cation, which is connected to the acid group through ionic bonds. It is something. The crosslinked quaternary nitrogen-containing water-soluble polymer is one obtained by crosslinking the above-mentioned quaternary nitrogen-containing water-soluble polymer.

Among the crosslinked quaternary nitrogen-containing water-soluble polymers, the most preferred is crosslinked cationic hydroxyalkylcellulose. When crosslinked cationic hydroquine alkylcellulose is used as a separation agent for biochemical substances, a higher separation effect can be obtained than when other crosslinked quaternary nitrogen-containing water-soluble polymers are used. In order to explain the present invention, a detailed explanation will be given using an example of crosslinked cationized hydroxyalkyl cellulose.

There are no particular limitations on the method for producing the crosslinked cationic hydroxyalkylcellulose used in the present invention. For example, in the method for producing reta-cationized hydroxyalkylcellulose shown in Japanese Patent Application No. 61-217672, the crosslinking agent is added at the time of adding the cationizing agent. If you add and react, the reactivity
It is advantageous in terms of economy.

Furthermore, as disclosed in JP-A No. 56-62801, cellulose ether is reacted with gly/nortrialkylammonium halide in a mixed solvent of water and a soluble organic solvent to obtain a cationic cellulose ether. , or may be used by crosslinking with a crosslinking agent.

Alkylene oxides used in the production of crosslinked cationic hydroxyalkylcellulose include ethylene oxide, propylene oxide, and various butene oxides.

By changing the amount of alkylene oxide added, hydroxyalkylcelluloses with various degrees of substitution can be obtained.

Depending on the type of alkylene oxide used, crosslinked products of cationic hydroxyethyl cellulose, cationic hydroxypropyl cellulose, and cationic hydroxybutyl cellulose can be obtained.

Among these, cross-linked cationic hydroxyethyl cellulose is most preferred because it is easy to handle and provides the highest separation effect. Further, as the cationizing agent used in the production of crosslinked cationic hydroxyalkylcellulose, glycitol trialkylammonium/.ride or its halohydride/type can be used.

Examples include glyciturtrimethylammonium chloride, glycinoltriethylammonium chloride, glyciturtrimethylammonium bromide, and 3-chloro-2-hydroquineprobyltrimethylammonium chloride.

Examples of crosslinking agents for cationized hydroxyalkylcellulose include epichlorohydrin, 1,3-fuchloro-2-
f-banol, 1,4-bis-(2,3゜propoxy)-butane, and the like.

Further, the amount of the crosslinking agent used is generally desirably about 1% to 20% by mole relative to the mole of glucose. If the amount of crosslinking agent used is outside this range, the separation performance will not be sufficient.

A method of utilizing a separation agent for biochemical substances selected from pyrotene, nucleic acids, and nucleic acids will be explained using an example of a method for separating nucleic acids using crosslinked cationic hydroxyalkylcellulose.

After adding the crosslinked cationic hydroxyalkyl cellulose obtained by the above-mentioned production method to an aqueous solution containing nucleic acid or a bacterial cell extract, the mixture is thoroughly stirred using a Stark or the like. Thereafter, it is allowed to stand still to precipitate the crosslinked cationic hydroxyalkylcellulose adsorbing the nucleic acid, thereby obtaining a supernatant liquid.

Alternatively, nucleic acids can be separated by separating crosslinked cationic hydroxyalkyl cellulose by filtration, centrifugation, etc. and obtaining a supernatant.

The amount of cross-linked cationized hydroxyalkyl cellulose to be added is adjusted depending on the nucleic acid content of the nucleic acid-containing solution to be removed, but in general, the number of moles of cationic groups added is equal to or more than the number of moles of nucleic acid. desirable.

The biochemical substance separation agent of the present invention can be used, for example, in the methods described above, but of course is not limited to the above methods. Another method of use is, for example, a commonly used method of filling a column with crosslinked cationic hydroxyalkyl cellulose.

In this case, when the solution containing the nucleic acid is passed over the cross-linked cationic hydroxyalkyl cellulose packed in the column, the effluent becomes a solution that does not contain the nucleic acid.

The case of separating nucleic acids has been explained in detail above, but how to use separation agents for these biochemical substances is as follows.
Exactly the same usage method can be applied even when separating nucleic acid-related substances and pyrozene.

〔Effect of the invention〕

As will be clear from the examples below, the biochemical substance separation agent of the present invention can separate biochemical substances selected from cyanoene, nucleic acids, and nucleic acid-related substances with sufficient efficiency just by -operation. Moreover, it has the excellent properties that other coexisting drugs are not subject to decomposition, and furthermore, no separation agent for biochemical substances remains in the solution after separation.

The reason why no biochemical substance separation agent remains in the solution after separation is because the crosslinked quaternary nitrogen-containing water-soluble polymer is sufficiently crosslinked and does not swell in the aqueous solution. However, it will not dissolve into the aqueous solution and remain in the aqueous solution.

The present invention will be explained below using 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 removing agent (product number C-7, Kurita Kogyo) using chitosan.

Add 1 cc of this aqueous solution to 0,
After adding to the solution dissolved in 15M NaC4,I Qcc, stir with a stirrer for 10 minutes. 5000 for the next 30 minutes
The precipitate was centrifuged at rpm, and the supernatant was collected. When the amount of nucleic acids contained in this supernatant was measured, 95% of the nucleic acids were removed.

However, when we measured the content of the nucleic acid removing agent remaining in this supernatant, we found that 3% of the original nucleic acid removing agent added to the nucleic acid solution
0% remained in the supernatant.

(Comparative Example 2) A 2% aqueous solution of cationized hydroquine ethyl cellulose (replacement of cationization agent! 1.5) using glycitultrimethylammonium chloride as a cationization agent was prepared. Add 1 cc of this aqueous solution to 1 m9 of calf trunk nucleic acid.
was added to a solution of 10 cc of 0.15 M NaCt, and stirred with a stirrer for 10 minutes.

Thereafter, the precipitate was centrifuged at 5000 rpm for 30 minutes, and the supernatant was collected. When the amount of nucleic acids contained in this supernatant was measured, 98% of the nucleic acids were removed.

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

(Example 1) A cross-linking agent was added to cationized hydroxyethyl cellulose (substitution degree of cationization agent: 1.1°, degree of substitution of hydroquinethyl group: 1.7) using glycitulutrimethylammonium chloride as a cationization agent. .3-Nokuroru 2
-7'/zonol 2% relative to the number of moles of glucose
In addition, it was reacted and crosslinked.

This cross-linked cationized hydroxyethyl cellulose 0
．． 19- to 0.15 MNa of calf body line nucleic acid 1m9
After adding Cl and l to the solution dissolved in Qcc, stir with a stirrer for 10 minutes. then 2000 rp for 10 minutes
The cross-linked cell was centrifuged at 500 m and the supernatant liquid was collected.

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

Moreover, no crosslinked cationized hydroxyethylcellulose was present in this supernatant.

(Example 2) Cationized hydroxyethyl cellulose using glycizoltrimethylammonium chloride as a cationizing agent (degree of substitution of cationizing agent 1.1°, degree of substitution of hydroxyethyl group 1.7) was added with epichlorohydrin as a crosslinking agent to glucose. 2% of the total number of moles was added and reacted to effect crosslinking.

This cross-linked cationization and droxyethyl cell
0.15' of calf body line nucleic acid 1m9
After adding it to the solution dissolved in aC4+ 10 CC, it was stirred with a stirrer for 10 minutes. then 2000 rp for 10 minutes
The cross-linked tube was centrifuged at m and the supernatant liquid was collected.

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

Moreover, no crosslinked cationized hydroxyethylcellulose was present in this supernatant.

(Example 3) Cationized hydroxyethyl cellulose using glycidyltrimethylammonium chloride as a cationizing agent (degree of substitution of cationizing agent: 1°1°, degree of substitution of hydroxyethyl group: 1.7) was treated with a crosslinking agent of 1.3 ~Cycloru 2
-7'Ropanol was added in an amount of 2% based on the number of moles of woculose to react and crosslink.

This cross-linked cationized hydroxyethyl cellulose 0
．． 154 was packed into a plastic mini-column, distilled water was poured from the top to wash it, and then a solution containing 1 μm of calf trunk nucleic acid dissolved in 10 cc of 0.15 M NaC was poured into the column, and the effluent was collected. . When the amount of nucleic acids contained in this effluent was measured, 99.5% of the nucleic acids were removed.

Moreover, no crosslinked cationized hydroxyethylcellulose was present in this effluent.

(Example 4) Cationized hydroxyethylcellulose using 3-chloro-2-hydroxy/f-ropyltrimethylammonium bromide as a cationizing agent (replacement of cationizing agent
,6. The degree of substitution of the hydroxyethyl group was 1.7), and 1,3-nochloro-2'-prono-2-nol, a crosslinking agent, was reacted at a ratio of 2% to the number of moles of glucose to effect crosslinking.

This cross-linked cationized hydroxyethyl cellulose 0
．． 11, calf thymus nucleic acid 1■ 0.15M NaCl,
After adding to the solution dissolved in 10 cc, stir with a stirrer for 10 minutes. Thereafter, the upper arm fluid was centrifuged at 2000 rpm for 10 minutes, and the upper arm fluid was collected.

When the amount of nucleic acids contained in the first liquid was measured, 96% of the nucleic acids were removed.

Moreover, no crosslinked cationized hydroquine ethylcellulose was present in this supernatant liquid.

Claims (4)

[Claims] (1) A separation agent for biochemical substances selected from pyrogens, nucleic acids, and nucleic acid-related substances, which is composed of a crosslinked quaternary nitrogen-containing water-soluble polymer. (2) The crosslinked quaternary nitrogen-containing water-soluble polymer is selected from pyrogens, nucleic acids, and nucleic acid-related substances as set forth in claim 1, characterized in that the crosslinked quaternary nitrogen-containing water-soluble polymer is crosslinked cationic hydroxyalkyl cellulose. Separation agent for biochemical substances. (3) The cross-linked quaternary nitrogen-containing water-soluble polymer is selected from the pyrogens, nucleic acids, and nucleic acid-related substances described in claim 1, characterized in that the cross-linked quaternary nitrogen-containing water-soluble polymer is cross-linked cationic hydroxyethyl cellulose. Separation agent for biochemical substances. (4) The cross-linked quaternary nitrogen-containing water-soluble polymer is a cross-linked cationic chitosan, wherein the cross-linked quaternary nitrogen-containing water-soluble polymer is selected from the group consisting of pyrogens, nucleic acids, and nucleic acid-related substances as set forth in claim 1. Chemical separation agent.