JPH05192175A - Production of diadenosine pllyphosphate solution - Google Patents

Abstract

PURPOSE:To obtain the subject material in high yield and purity by treating a solution containing diadenosine polyphosphate (ApnA) with a porous synthetic adsorbent. CONSTITUTION:Styrene and divinyl benzene are polymerized, changed surface polarity and introduced electron attractive groups to obtain a porous synthetic adsorbent (A). A fermented liquid (B) containing the component A and ApnA and mixed with 1-100 times of volume of the component B to the component A is stirred at pH1-5 to obtain an adsorbed material (C) specifically adsorbed solely ApnA on the component A. After the component C is washed with alcohol, etc., ApnA is extracted by mixing and stirring with an eluate such as 2-5 times amount of ethanol, etc., and filtered and separated to produce highly pure diadenosine polyphosphoric acid solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a solution containing diadenosine polyphosphate (hereinafter abbreviated as ApnA) in high purity. More specifically, high purity A
The present invention relates to an industrially advantageous method for producing a pnA solution in high yield.

[0002]

2. Description of the Related Art ApnA acts on DNA synthesis,
It has various physiological effects such as effects on blood coagulation and effects on blood vessels (J. Luthje, Klinische Wochenschrift 67, 317-327 (1989)), and is used as a drug and a drug raw material. Is a substance that is expected to be used.

There are two main methods for obtaining this ApnA.
It is produced by the reaction of condensing the molecule adenosine polyphosphate with an enzyme, but the unreacted adenosine polyphosphate (adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP)) is present in the reaction solution of ApnA. , Adenosine tetraphosphate (ppppA), etc.] and impurities such as proteins derived from enzymes, and is qualified as a pharmaceutical product.
It is essential to prepare a high-purity ApnA solution by preparing some kind of purification means in this reaction solution in order to produce the above.

[0004] Conventionally, a high-purity ApnA reaction liquid has been used.
As a method for producing the solution A, a basic ion exchange resin or the like is used to ionically adsorb ApnA to the ion exchange resin, and then the salt concentration of the eluate is changed stepwise to elute ApnA. , A method for producing a solution containing ApnA in high purity is known [JR Reiss and JGMoffa (JR Reiss and JGMoffa
tt) Journal of Organic Chemistry
(J.Org.Chem.) 30,3381-3387 (1965)].

[0005]

In the above conventional method, the exchange capacity of the basic ion exchange resin is originally low, and impurities such as adenosine polyphosphate and enzyme contained in the fermentation liquid are also adsorbed. , The amount of adsorption of ApnA on the resin decreases. In addition, since the degree of separation of ApnA from other impurities is low during elution, it was extremely difficult to obtain high-purity ApnA in high yield, especially on an industrial scale, in terms of cost.

The present invention selectively adsorbs ApnA on a resin to increase the amount of ApnA adsorbed and to increase ApnA.
The purpose is to easily obtain a solution containing A in high purity.

[0007]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that a porous synthetic adsorbent can be used and reached the present invention.

That is, the present invention provides a method for producing an ApnA solution characterized in that a solution containing ApnA is brought into contact with a porous synthetic adsorbent to specifically adsorb ApnA, and then the adsorbed ApnA is eluted. It is a summary.

[0009] The porous synthetic adsorbent, which was conventionally considered to be a resin for industrial use, could be subjected to delicate separation of an ultrahigh-purity (99.7% or more) product that can be used as a drug, which is the object of this purification. That is quite amazing. The present invention is the first example in which a porous synthetic adsorbent is used for purification of ApnA.

The present invention will be described in detail below. In the present invention, it is first necessary to obtain an ApnA reaction solution. As a method therefor, a method using an aminoacyl-tRNA synthetase and using ATP as a raw material [Molecular and
Cellular Biochemistry (Mol.Cell.Biochem.)
, 52, pp. 3 to 11 (1983), described by S. Blanket et al.].

In the ApnA reaction solution obtained by the above method, the reaction product ApnA and unreacted AMP,
It includes ADP, ATP, ppppA and the like. In addition, the enzyme and the protein derived therefrom may be contained as they are.

In the production method of the present invention, the above-mentioned ApnA fermentation broth may be used as it is, or the fermentation broth deproteinized with a membrane, acid, alkali or the like may be used.

As the porous synthetic adsorbent used in the present invention, those which are usually produced by polymerizing styrene and divinylbenzene are preferable, and those whose surface polarity is changed, especially those into which an electron withdrawing group is introduced are desirable. For example, Sepa beads SP
What is marketed as a series (made by Mitsubishi Kasei) may be used.

In the production method of the present invention, the fermentation liquid containing ApnA is brought into contact with the porous synthetic adsorbent to specifically adsorb only ApnA. As a method of making this contact,
Examples thereof include a batch method in which a fermentation solution and a porous synthetic adsorbent are directly stirred, and a column method in which a porous synthetic adsorbent is packed in a column and the reaction solution is passed. Whichever method is used, the effect is not so different.

In the case of the column method, for example, the following can be performed. After the column is filled with the porous adsorbent, the ApnA reaction liquid is passed to selectively adsorb only ApnA to the porous adsorbent. This porous adsorbent is washed with a solution containing no organic solvent such as alcohol to wash away impurities, and then ApnA is eluted with a solution containing alcohol to obtain a high purity solution.

The flow rate is not particularly limited because the amount of adsorption is not significantly affected by the flow rate, but it is preferable that the volume is around 3 column volumes per hour, which is efficient and preferable, but a flow rate higher than this is used. May be. The pH of the reaction solution is not particularly limited because it is not significantly affected by the adsorption of ApnA on the synthetic adsorbent, but is not particularly limited.
It is desirable to be on the acidic side, more preferably pH 1-5
Is.

After the adsorption of ApnA, pure water, a buffer solution,
A synthetic adsorbent is washed by passing a solution containing no organic solvent such as hydrochloric acid diluted solution, especially alcohols, through the column. For example, water, saline, or a buffer solution of phosphoric acid, imidazole, hepes, tris, acetic acid, glycine or the like can be used. When using a buffer, the concentration or pH of the buffer
It is not particularly limited to. The volume of cleaning liquid used for cleaning is
The amount is sufficient as long as the impurities are sufficiently washed away, and usually the same amount as the volume of the adsorbent used or more, preferably 3 times or more is used. The cleaning liquid contains impurities such as AMP, ADP, and A.
TP, protein, etc. are included.

Next, ApnA is eluted from the adsorbent.
An organic solvent is used for elution of ApnA from the adsorbent. Although there is no great difference in the effect of using any organic solvent, alcohols are preferable, and an alcohol solution diluted to about 1 to several% can be sufficiently eluted. For example, a solution of ethanol, methanol, n-propanol, isopropanol, glycerin, ethylene glycol or the like is used. The volume of the eluent used for elution may be any amount as long as ApnA is sufficiently eluted, and the volume is usually the same as or more than the volume of the adsorbent used, preferably 1 to 3 times.

When the batch method is used, the following can be carried out. In a suitable container, the porous synthetic adsorbent and the ApnA reaction solution are thoroughly mixed and stirred, and then ApnA is mixed.
Only adsorb the adsorbent. At this time, the volume of the ApnA reaction solution used may be any number of times the volume of the adsorbent, and is usually 1 to 100 times, preferably 1 to 10 times. The pH of the reaction solution is not particularly limited, but it is preferably acidic, and adsorption is usually performed in the range of pH 1-5.

After the adsorption, the adsorbent and the reaction solution are separated by filtration or the like. This adsorbent is mixed and stirred with the washing liquid described in the column method to wash impurities. At this time, the volume of the cleaning liquid used may be any amount as long as the impurities can be sufficiently cleaned, and normally, the amount is 3 times or more the volume of the adsorbent.
It is also possible to perform the washing a plurality of times. After the washing, the adsorbent and the washing liquid are separated by filtration or the like. This adsorbent is mixed and stirred with the eluent described in the column method to elute ApnA. In this case, the volume of the eluent may be any amount, and practically, the amount is 2 to 5 times the volume of the adsorbent. After elution, the adsorbent and the eluate are separated by filtration or the like to obtain a solution containing ApnA in high purity.

[0021]

EXAMPLES The present invention will now be described in detail with reference to typical examples, but it should not be understood that the present invention is limited thereby. Example 1 In order to obtain a solution containing diadenosine tetraphosphate (Ap4A) in high purity by the batch method, first, M. Kawakami (M.
FEBSletter by Kawakami) et al.
s) 185, 162-164 (1985), Escherichia coli JE5506-pkD15 self-cloned with isoleucyl-tRNA synthetase
11950), pre-treatment of nucleic acids and the like, followed by purification by DEAE-Sepharose and hydroxyapatite column chromatography, and using isoleucyl-tRNA synthetase that became electrophoretically almost single, A. Brevet (A. Brevet ) Et al. Proceeding of the National Academy of Sciences (Proc.Nat
l.Acad.Sci, USA) Vol. 86, pages 8275-8279 (19)
89), 15 mM ATP, 3
0 mM magnesium chloride, 3 mM zinc chloride and 3 m
The reaction was carried out in 100 mM Tris-HCl buffer (pH 7.8) containing M isoleucine to obtain a reaction solution of Ap4A.

Ap4A reaction solution 6 thus obtained
After removing 00 ml by filtering impurities, it was thoroughly stirred with 100 ml of Sepa beads (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). After separating the Sepa beads and the reaction solution by filtration, the Sepa beads were treated with 1800 ml of 50 mM Hepes buffer (pH
It was mixed with 7.5), stirred, and washed. After separating the Sepa beads and the washing solution by filtration, the Sepa beads are mixed with 200
Ap4A was eluted by mixing with 20 ml of 20% ethanol and stirring, and Sepa beads and the eluate were separated by filtration.
A solution containing A in high purity was obtained.

The reaction solution, the washing solution and the eluate are respectively described in A. Ogilvie et al., Analytical Biochemistry (Anal. Biochem.), Vol. 134, 382-392 (1983). Thus, using a NOVA-Pac C-18 column (manufactured by Waters), the components were analyzed by high performance liquid chromatography using a 30 mM phosphate buffer (pH 7.0) as a mobile phase.

[0024]

[Table 1]

From this result, Ap4A was obtained by the batch method.
It was shown that only the solution was adsorbed on Sepabeads, and by elution with ethanol, a solution containing Ap4A in high purity could be produced with a high recovery rate.

Example 2 Ap4A reaction solution 100 obtained by the method shown in Example 1
After insoluble matter was removed by filtration, ml was passed through a column (size 70 mmΦ × 250 mm) loaded with 1 l of Sepa beads and thoroughly washed with distilled water, and then eluted with a gradient of 0% to 50% ethanol concentration. I did it. The results are shown in Table 2.

[0027]

[Table 2]

From these results, Ap4A in the column method
It was shown that only the solution was adsorbed on Sepa beads, and a solution containing Ap4A in high purity could be produced with high recovery rate by gradient elution with ethanol.

Example 3 Ap4A reaction solution 100 obtained by the method shown in Example 1
After removing insoluble matter by filtration, 150 l of Sepa beads
The solution was passed through a column (size 44 cmΦ × 100 cm) on which was loaded, was thoroughly washed with a 0.01 N-HCl solution, and was then eluted with a 0.01 N-HCl solution of 1% ethanol. The results are shown in Table 3.

[0030]

[Table 3]

From these results, Ap4A in the column method was used.
It was shown that only the solution was adsorbed on Sepa beads, and the solution containing Ap4A in high purity could be produced with high recovery rate by isocratic elution with ethanol.

Comparative Example 1 Ap4A reaction solution 1.2 obtained by the method shown in Example 1
After removing insoluble matter by filtration, 1 DEAE-Sepharose Fast Flow 12
The solution was passed through a column (size 18 cmΦ × 60 cm) loaded with 1 to elute with a NaCl gradient.
The Ap4A elution fraction (three fractions) was subjected to component analysis.
The results are shown in Table 4.

[0033]

[Table 4]

From these results, the conventional method using DEAE-sepharose requires about 10 times the amount of resin to purify the same amount of Ap4A as the method using sepa beads described in the present invention. In addition, a buffer solution or N
Since aCl is used, processing after elution is extremely difficult. From this, the effectiveness of the method using sepa beads described in the present invention was shown.

Example 4 Using an isoleucyl-tRNA synthetase obtained by the same method as in Example 1, and also referring to Example 1, 5 mM AT was used.
P, 10 mM ADP, 30 mM magnesium chloride,
Containing 3 mM zinc chloride and 3 mM isoleucine 10
The reaction was performed with 0 mM Tris-HCl buffer (pH 7.8) to obtain a reaction solution of Ap3A. 100 ml of the obtained Ap3A reaction solution was filtered to remove insoluble matter, and then passed through a column loaded with 1 l of Sepa beads, washed with 500 ml of distilled water and eluted with 300 ml of 5% ethanol. Result is fifth
Shown in the table.

[0036]

[Table 5]

From these results, it was shown that a solution containing Ap3A in high purity can be produced with a high recovery rate.

Example 5 Using an isoleucyl-tRNA synthetase obtained in the same manner as in Example 1, and also referring to Example 1, 5 mM AT was used.
P, 10 mM ppppA (Ap4), 30 mM magnesium chloride, 3 mM zinc chloride and 3 mM isoleucine in 100 mM Tris-HCl buffer (pH 7.
The reaction was performed in 8) to obtain a reaction solution of Ap5A. 100 ml of the obtained Ap5A reaction solution was filtered to remove insoluble materials, and then passed through a column loaded with 1 l of Sepa beads, washed with 500 ml of distilled water and eluted with 300 ml of 2% ethanol. The results are shown in Table 6.

[0039]

[Table 6]

From these results, it was shown that a solution containing Ap5A in high purity can be produced with a high recovery rate.

[0041]

According to the present invention, the target adsorption amount of ApnA is as large as several times to several tens of times that of the conventional method, and Ap
Since only nA is selectively adsorbed, high-purity Apn can be easily obtained.
Solution A can be produced in high yield.

Claims (1)

[Claims] 1. A method comprising contacting a solution containing diadenosine polyphosphate with a porous synthetic adsorbent to specifically adsorb diadenosine polyphosphate, and then eluting the adsorbed diadenosine polyphosphate. Preparation of diadenosine polyphosphate solution.