JPS5971687A - Stable composition containing glucose-6-phosphate dehydrogenase - Google Patents

Abstract

PURPOSE:To provide a composition containing glucose 6-phosphate dehydrogenase, having excellent storage stability, and composed of an aqueous composition or its freeze-dried product containing glucose 6-phosphate dehydrogenase and an aminopolycarboxylic acid or its salt. CONSTITUTION:An aqueous composition containing glucose 6-phosphate dehydrogenase and an aminopolycarboxylic acid and/or its salt, or freeze-dried product of the above composition. The glucose 6-phosphate dehydrogenase may be of isolated state or a state bonded with an immunoactivating substance such as hapten, antigen, antibody, etc. The hapten and antigen are e.g. an antiepileptic such as phenobarbital, a cardiotonic such as digoxin, and an antibiotic such as gentamicin. The amount of the aminopolycarboxylic acid in the enzyme solution is preferably >=0.1W/V% for freeze-dried product, and >=5W/V% for solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to stable glucose-6 monophosphate dehydrogenase proprietary compositions. More specifically, the present invention relates to a glucose-6-phosphate dehydrogenase-containing composition that has extremely excellent storage stability.

Glucose-6-phosphate dehydrogenase (hereinafter abbreviated as 06PDH) is contained in yeast, bacteria, and various mammalian tissues, and its production methods include, for example, extraction of M from Tetraiconostoc mesente poides, or Methods for extraction from Acetobacter suboxidans, yeast, etc. have been established.

G6PDH is involved in the primary reaction in vivo, as is well known, and is involved in the primary reaction of nicotinamide adenine dinucleotide phosphate (NA).
DP), used for the determination of glucose-6-phosphate.

Furthermore, in combination with other enzymes, it is widely used in clinical testing departments, such as in the measurement of 16 glucose-hair-phosphate and in vivo enzyme activity.

D-glucose-6-1J acid + MAD(P)”6PD
H □D-glucono δ-lactone-6-phosphate ten NAD (
p)H This G6PDH is said to be relatively stable under certain special conditions, such as in an ammonium sulfate suspension at a high enzyme concentration, in an S50% glycerol aqueous solution, or in a freeze-dried state. In addition to ammonium sulfate, other polyvalent anionic substances with high ionic strength, such as inorganic sulfates and phosphates, are known as substances that stabilize enzymes in suspension. However, when an enzyme is stabilized under conditions of concentrated enzyme as described above, the highly concentrated enzyme must be diluted when the enzyme is used. For this reason, 1. For example, in the case of a lyophilized product, the necessary amount is weighed and used, but this operation is complicated and tends to lack accuracy. Enzyme solutions are prone to problems with their storage stability.

In addition, in the case of enzyme suspensions, the inorganic salts used to stabilize the enzymes are highly concentrated and tend to inhibit the enzyme reaction. Usually, the substance is dissolved in a buffer solution, subjected to one dialysis, and desalted before use. The use of enzymes stabilized under concentrated conditions as described above has the disadvantage that the operation is extremely complicated.

Furthermore, 06PDH can be present in dilute concentrations either in the free state or in the bound state (eg, as an immunoactive substance). Furthermore, if the suspension is to be stabilized, there are drawbacks such as the necessity of desalting treatment as described above in order to avoid adverse effects on enzyme activity.

The present inventors have conducted extensive studies in view of these shortcomings, and have developed a stabilizer that does not have any adverse effect on the reaction system of free or bound G, 6FDH, and has extremely excellent storage stability. They discovered aminopolycarboxylic acid and completed the present invention.

That is, one aspect of the present invention is a stable ()6pDH-containing composition comprising an aqueous composition or a lyophilized product containing G6PDH and an aminopolycarboxylic acid and/or a salt thereof.

The present invention is significantly different from conventional ammonium sulfate or sodium sulfate suspensions in that even if a particularly high concentration of aminopolycarboxylic acid or its salt is added, the enzyme reaction system is not adversely affected at all.

The G6PDH used in the present invention may be of any origin such as yeast, bacteria, and various animal tissues; for example, Leuconostoc mesenteroides (Leuco
nostock mesJ(enteroides L
Escherichia coli (Fj day cherichia coli),
Pseudomonas pseudomonas
nas 5 acaharophila) H Acetobacter suboxidans (Aoetobactsr.
A free enzyme is used which is extracted and purified by a conventional method after culturing microorganisms such as 5ubox%ane).

06PDH may be in a free state or in a bound state bound to an immunoactive substance) Examples of the immunoactive substance include hapten,
Examples include antigens and antibodies.

Haftene and antigens include steroids, vitamins) alkaloids (e.g. morphine, heroin, etc.), amino acids, polypeptides, catecholamines, xanthines, arylalkylamines, 1 heterocycles, etc. Brokerage 1 Furthermore, hormones (growth hormone,
Thyroid hormone (such as MJillffi hormone), protein (engineering f
G, As σ-fetoprotein 1 Protein A
1 laminin, phipronectin, erythroboetin, etc.), and typical examples include antiepileptic drugs such as fuemevalpital and phenytoin, cardiotonic drugs such as digoxin and lidocaine, and antibiotics such as gentamicin. . As the antibody, used is Antibody 1, such as an anti-insulin antibody, which is obtained by immunizing a mammal such as a goat or a sheep with the above-mentioned hapten or antigen using a conventionally known method. Furthermore, a covalent bond is used as a form of bond between G6PDK and an immunoactive substance.

As the aminopolycarboxylic acid, ethylenediamine-4
Acetic acid (hereinafter abbreviated as KDTA), 1,2-di-hexanediamine-4-acetic acid (hereinafter abbreviated as 0DTA), dihydroxyethylglycine, diamine/propanol-4-acetic acid 1-diethylenetriamine-5-acetic acid (hereinafter referred to as DTPA). omitted)
, ethylenediamine orthohydroxyphenylacetic acid, ethylenediamine-2-acetic acid, glycol ether diamine-4-acetic acid to hydroxyethylethylenediamine-3
- Acetic acid, ethylenediamine-2-propionic acid, hydroxyethylimino-2-acetic acid, imino-2-acetic acid, diaminopropane-4-acetic acid, nitrilo-2-acetic acid/propionic acid, nitrilo-3-acetic acid (hereinafter referred to as NTA )
, nitrilopropionic acid, triethylenetetramine-6
-acetic acid etc., and its salts include its metal salts,
Examples include ammonium salts, and each may be used alone or in combination. Particularly preferred aminopolycarboxylic acids include EDTA, 0I)TA, and the like.

In addition, the aminopolycarboxylic acid has a ratio of 0.1 weight to volume (W/% or more is preferable in the case of 1 freeze-drying per enzyme solution)
In the case of a solution, 5 w/v% or more is desirable. If the amount added is more than 40 w/v%, the solubility will be poor, making it unsuitable for practical use. On the other hand, if it is less than 0° o 1 w/v%, the stabilizing effect of the enzyme will be poor. is small.

The aqueous composition containing the above-mentioned additives has a bond of 5 to 9, preferably around 6 to 8. If adjustment is necessary depending on the type of aminopolycarboxylic acid or the amount added, it is carried out by an appropriate method. Any buffer that is commonly used within the above pH range can be used; particularly preferred buffers include triethanolamine-11, lis(hydroxyethyl)aminomethane-hydrochloric acid, and various types of buffers. 1. It is not limited to these, and the concentration can be used within a practical concentration range.

In this way, the aqueous composition containing G(IPDH and aminopolycarboxylic acid or its salt) may be in the form of an aqueous solution as it is, or may be further dried by some drying means including freeze-drying.

When obtaining an enzyme-containing composition by some drying process 1
In particular, it is possible to add excipients to prevent quality deterioration due to aging, transportation, etc. in vials, and to facilitate dissolution before use.Excipients include lactose, saccharose, dextrin, β-cyclodextrin, Examples include dextran and its derivatives, chondroitin sulfate, sugars such as starch, polyethylene glycols, polyvinylpyrrolidone, proteins such as albumin, and other polymeric substances.The added concentration is O.1 w/v% or more, 30 W/v. v
Used below %.

Deterioration over time during the storage period of the enzyme-containing composition of the present invention is almost completely prevented even at 40°C for one month. Furthermore, even when the aminopolycarbonate vinegar in the enzyme-containing composition is used at a high concentration, It will not cause a decrease in enzyme activity. That is, the enzyme-containing composition according to the present invention is extremely stable even under conditions where the enzyme is diluted, and is a composition that is impractical due to its ease of use. The enzyme-containing composition of the present invention is used as a clinical test reagent, including a diagnostic reagent for enzyme immunoassay.

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

Example I G6PDl ((manufactured by Toyobo Co., Ltd., 500U/Ill+) 0.
5gv was dissolved in 100mM) lis(hydroxymethyl)aminomethane/hydrochloric acid buffer (pH 7°9) 125- to prepare a 2U/14 G6PDH stock solution. This stock solution 1 containing 20% EDTA-2N & previously prepared
o OmM) Lis-HCl buffer (pl(7,9)20
m/d and mixed to make the 06PDH-containing aqueous composition WM.
Manufactured. Next, put the composition in a tightly closed glass container,
It was left in the dark in an incubator at 40°C for one month. Next, 0.5 enl of the aqueous composition was added and mixed with 0.2 ml of 100 mM madanesium chloride, 35 mM glucose, 6.
Absorbance change in (Δ0D340 in/min)
was measured. The results are shown in Table 1.

Table 1 shows the case where 0DTA-2Na, additive-free, ammonium sulfate, sodium sulfate, sodium acetate, and sodium citrate were used instead of EDTA-2Na.

1) The case without additives is set as 100%.

2) The activity immediately after preparation is 100%; 3) The activity immediately after preparation is 100%.

As is clear from Table 1, the additives used in the present invention show no decrease in enzyme activity even when added at high concentrations, have excellent storage stability, and exhibit G6PDH significantly superior to any of the controls. Containing composition.

Example 2 N-carboxymethylphenobarbital was synthesized by reacting phenobarbital with methyl chloroacetate and hydrolyzing it.
A phenobarbital labeled G06 P D H aqueous composition prepared by blending the additives shown in Table 2 combined with the G6PDH aqueous composition of Example Zhu 1 was used in place of the G6 PDH aqueous composition of Example 1. Residual enzyme activity was measured under the same conditions as in Example 1. The results are shown in Table 2.

Furthermore, enzyme immunoactivity was also measured using this composition. That is, fumebarbital-labeled G6PDH immediately after preparation
l− (OOmM) Lis-HCl buffer (p+ (7,90
) 2 m, phenobarbital standard solution (30μy7n
t) 2. After heating at S37°C x 3 molecules, 50 μg of previously prepared fumebarbital antibody (an antibody obtained from sheep by binding fumebarbital to bovine serum albumin and using it as an antigen) was added to the antigen antibody. Changes in enzyme activity due to the reaction were measured using ΔOD3407mn. FIG. 1 shows the case where IDTA-2Nawk Bet ammonium and sodium sulfate are used. In the figure, the horizontal axis represents the content of the additive, and the vertical axis represents the enzyme activity (%) when the additive is contained, with the enzyme activity when the additive is not contained as 100%.

As is clear from FIG. 1, KDTA used in the present invention
Even when -22Ja is added at a high concentration, no decrease in enzyme activity is observed. Moreover, as seen in Table 2, the aqueous compositions according to the invention exhibit better storage stability than any of the controls.

Table 2 Modification 1) Immediately after preparation - Enzyme activity without additives is assumed to be 100% 14
The residual activity after 2 weeks at 0°C is shown.

Example 3 G6PDH aqueous solution prepared in the same manner as in Example 1 and phenol rubital labeled 06 prepared in the same manner as in Example 2
Each PDH aqueous solution 1- was prepared in advance with 2% FliDTA-
100mM) Lis-HCl buffer containing 3K et
(pH 7,9) and then a freeze-dried product was obtained. 40'CX 1'r Moon N Dark F
After being left in the Jr N incubator, Example 1,
The storage stability of the sample Aoigumi v.1 was evaluated under the conditions described in 2.

For comparison, an enzyme-containing composition containing various additives was also tested at the same time (the pH was adjusted to 7.9). The results are shown in Table 3.

As is clear from Table 3, the enzyme-containing lyophilized product according to the present invention has significantly better stability than the control composition.

Example 4 Lactose and manrolidone were each added at 2% as excipients to the composition of Example 1, and freeze-dried to obtain a freeze-dried product.

Next, the results were evaluated in exactly the same manner as in Example 1, and it was confirmed that the shape stability was excellent, and the storage stability was also extremely poor.

[Brief explanation of the drawing]

FIG. 1 shows changes in enzyme activity due to antigen-antibody reactions using the phenobarbital-labeled 06PDH aqueous composition of the present invention. The horizontal axis shows the concentration (%) of the additive, and the vertical axis shows the ()6 PDH activity ratio (%) associated with the antigen-antibody reaction. In the figure, 1 is E
DTA-2NaS2 is ammonium sulfate, and 3 is sodium sulfate. Patent applicant: Toyobo Co., Ltd. 2 Procedural amendment (voluntary) December 23, 1980 Commissioner of the Patent Office Kazuo Wakasugi 1 Indication of case 1981 Patent Application No. 179632 2 Completion of invention, title Stable glucose-6-phosphate dehydrogenase-containing composition δ Relationship with the case of a person who failed to make an amendment Patent applicant 2-2-8-4 Dojimahama, Kita-ku, Osaka City “Patent claim” in the specification subject to amendment "Scope of the Invention" column and "HI3 of the Invention"
Column 5 of "Detailed Explanation", Contents of Amendment (1) The scope of claims will be corrected as shown in the attached sheet. (2) From the end of page 7 to the second line of page 8 of the specification: “If the amount added exceeds 40W/4%, the solubility will be poor.
[The addition amount is generally 40W/v% or less, which is a practically preferable range from the viewpoint of solubility, but depending on the type of 1-aminopolycarboxylate, it may be 40V7/v%.
There are some compounds that have good solubility even at the above concentrations, but the concentration is not limited to these. ” he corrected. (3) On page 1O, line 12 of the same page, after “0.5-”, add “100mM triethanolamine/loamic acid buffer (pH 7+9) 2+lom7.11 W
+ M NADP 0.1-1". Attachment Claim (1) Stable glucose-6-composed of an aqueous composition or freeze-dried product containing glucose-6-phosphate dehydrogenase and an aminopolycarboxylic acid and/or a salt thereof
A composition containing phosphate dehydrogenase. (2) The stable glucose-6-phosphate dehydrogenase according to claim 1, wherein the glucose-6-phosphate dehydrogenase is glucose-6-phosphate dehydrogenase in a free state or in a bound state. Composition containing phosphoric acid dehydrated stimulant. (3) Glucose-6-phosphorus dehydrogenation u k in the bound state
Claim 1, characterized in that it is a conjugate of caculcose-6-phosphate dehydrogenase and an immunoactive substance.
Stable glucose-6 monophosphate dehydrogenase-containing composition 1゜(4) as described in Section 1, characterized in that it contains 0.1 to 40 W/V% of aminopolycarboxylic acid and/or its salt in the aqueous composition. A stable glucose-6-phosphate dehydrogenase-containing composition according to claim 1.

Claims (1)

[Scope of Claims] (1) A stable glucose-6-phosphate comprising an aqueous composition or a lyophilizate containing glucose-6-phosphate dehydrogenase and an aminopolycarboxylic acid and/or a salt thereof.
A composition containing phosphate dehydrogenase. (2) The stable glucose-6-phosphate dehydrogenase according to claim 1, wherein the glucose-6-phosphate dehydrogenase is glucose-6-phosphate dehydrogenase in a free state or in a bound state. A composition containing phosphate dehydrogenase. (8) A patent characterized in that the bound glucose-6-phosphate dehydrogenase is a conjugate of glucose-6-phosphate dehydrogenase and an immunoactive substance (4) Aminopolycarboxylic acid and/or or its salt in an aqueous composition.
The stable glucose-6-phosphate dehydrogenase-containing composition according to claim 1, which contains 61 to 40 w/v% gold.