JP4557571B2 - Method for stabilizing fructosyl peptide oxidase - Google Patents

Description

  The present invention relates to a method for stabilizing a fructosyl peptide oxidase (hereinafter referred to as “FPOX”) lyophilized preparation used for clinical diagnosis, and a stabilized FPOX lyophilized preparation.

  A glycated protein is a protein in which the protein is non-enzymatically glycated, and the aldehyde group on the sugar, that is, aldose (monosaccharide and derivate having a potential aldehyde group) side, and the amino group on the protein side are non-enzymatic. It is generated as a result of covalent bonding to. Examples of the amino group of the protein include the α-amino group of the N-terminal amino acid and the ε-amino group of the internal lysine residue side chain. These glycated proteins are also called so-called Amadori compounds because the Schiff base generated as a reaction intermediate undergoes Amadori transfer.

  Glycated proteins are contained in body fluids such as blood in living bodies and biological samples such as hair. The concentration of glycated protein present in blood strongly depends on the concentration of saccharides such as glucose dissolved in serum. In diabetic state, the production of glycated proteins is enhanced, and the concentration of glycated hemoglobin in serum and glycated albumin in serum reflects the average blood glucose level over a certain period of time. Measuring is important for diagnosis and management of symptoms of diabetes.

Conventional methods for quantifying glycated proteins include, for example, a method using high performance liquid chromatography (see, for example, Non-Patent Document 1), and a method using a column packed with a solid to which boric acid is bound (see, for example, Non-Patent Document 2). , A method using electrophoresis (for example, see Non-patent Document 3), a method using an antigen-antibody reaction (for example, refer to Non-Patent Document 4), and a method for colorimetric determination of reducing ability using a tetrazolium salt (for example, non-Patent Document 4) Patent Document 5), a method for colorimetric determination after oxidation using thiobarbituric acid (for example, see Non-Patent Document 6), a method using an enzyme such as glycated amino acid oxidase (for example, see Patent Documents 1 to 16), etc. It has been known. Recently, a new method for measuring glycated protein has been disclosed as a method for measuring glycated protein more accurately than the above method (see, for example, Patent Document 17). In this measurement method, a sample containing a glycated protein is treated with a protease, the fructosyl peptide is released from the glycated protein, FPOX is allowed to act on the released fructosyl peptide, and the product is measured to measure the glycated protein. Therefore, it is attracting attention as a highly accurate measurement method with a short time and simple operation.

  In order to keep the enzyme stable for a long period of time during storage and transportation, the enzyme is often lyophilized to form a preparation. When lyophilization is performed, it is generally well known that an enzyme is stabilized by adding a sugar such as glucose to the enzyme solution. However, when the enzyme is an enzyme using a glycated product such as FPOX as a substrate, The use of is not appropriate. This is because the sugar added during freeze-drying causes an Amadori reaction with amino acids, peptides, proteins, etc. that coexist in the reaction system, producing glycated products, which causes an increase in the background when measuring glycated proteins. Because. For these reasons, it is considered that sugar is not suitable as a stabilizer for freeze-drying FPOX, but a method for stabilizing FPOX using a substance other than sugar is not known.

Japanese Patent Publication No. 05-33997 JP-A-11-127895 WO 97/13872 pamphlet Japanese Patent Publication No. 06-65300 Japanese Patent Laid-Open No. 02-195900 Japanese Patent Laid-Open No. 03-155780 Japanese Patent Laid-Open No. 04-4874 JP 05-192193 A Japanese Patent Laid-Open No. 06-46846 JP-A-11-155596 JP 10-313893 A Japanese Patent Laid-Open No. 11-504808 JP 2000-333696 A JP 2001-54398 A JP 2001-204495 A JP 2001-204494 A JP 2001-95598 A Cole RA, 3 others, “A rapid method for the determination of glycosylated hemoglobins using high pressure liquid chromatography”, Metabolism, 1978 5 Moon, Vol. 27, No. 3, p.289-301 Klenk DC, 9 others, “Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric: Affinity chromatography: quantification of glycosylated hemoglobin by affinity chromatography: comparison of colorimetric and ion exchange methods and ion-exchange methods, and effects of common interferences) ", Clin Chem, October 1982, Vol. 28, No. 10, p. 2088-2094. Menard L, 5 others, “Quantitative determination of glycosylated hemoglobin A1 by agar gel electrophoresis”, Clinical Chemistry (Clin Chem), October 1980, Vol. 28, No. 11, pp. 1598-1602 Journal of the Japan Society for Clinical Laboratory Automation, 1993, Vol. 18, p.620 Johnson RN, two others, “Fructosamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control”, Clinica Clin Chim Acta, January 7, 1983, Vol. 127, No. 1, p. 87-95 Dolhofer R, 1 other, “Improvement of the thiobarbituric acid assay for serum glycosylprotein determination”, Clin Chim Acta May 5, 1981, Vol. 112, No. 2, p. 197-204

  An object of the present invention is to provide a method for stabilizing a FPOX lyophilized formulation and a stabilized lyophilized formulation of FPOX.

  As a result of intensive studies to solve the above problems, the present inventors have made the stability of the FPOX lyophilized preparation extremely improved by allowing a chelating reagent, sugar alcohols, amino acids, and metal salts to coexist. Based on these findings, the present invention has been completed.

  That is, the present invention relates to a method for stabilizing a FPOX freeze-dried preparation, wherein one or more reagents selected from chelating reagents, sugar alcohols, amino acids, and metal salts are allowed to coexist with FPOX. It further relates to a stabilized FPOX lyophilized formulation obtained by this stabilization method.

  According to the method of the present invention, a FPOX lyophilized preparation can be stabilized by allowing a chelating reagent, sugar alcohols, amino acids, and metal salts to coexist. Using this stabilization method, a stabilized lyophilized formulation of FPOX can be produced in a simple manner and inexpensively.

  Hereinafter, the present invention will be described in detail. FPOX used in the present invention is an oxidase that acts on fructosyl peptide in the presence of oxygen to produce α-ketoaldehyde, peptide, and hydrogen peroxide, and any oxidase is included as long as it has this action. . The origin of FPOX used in the present invention may be any, for example, filamentous fungi, yeast, actinomycetes, bacteria, archaea, etc., but is not particularly limited, but preferably, for example, Coniochaeta, Eupenicillium ( FPOX originating from Eupenicillium), Achaetomiella, etc. These FPOXs can also be those produced by gene recombinants.

  In the present invention, the FPOX lyophilized preparation refers to a powdered enzyme preparation obtained by lyophilizing the above FPOX using a lyophilizer generally used and pulverizing the obtained dried product. The freeze-drying of FPOX is performed using, for example, a vacuum freeze-dryer (FREEZVAC-1 CM type) manufactured by Tozai Tsusho Corporation. A tray for storing FPOX is placed on the drying shelf of this vacuum freeze dryer and cooled to -50 ° C. in advance. 300 ml of FPOX (Kikkoman) is aseptically filtered through a membrane filter and then poured into the tray. . The sample is frozen (24 hours) while maintaining the temperature in the vacuum freeze dryer at -50 ° C. When the sample is frozen, the cold trap of the vacuum freeze dryer is cooled to −50 ° C., and after 30 minutes, the vacuum pump is operated and drying is started at a vacuum degree of 4.54 PASCAL. 24 hours after the start of drying, the shelf temperature of the drying shelf of the vacuum freeze dryer is set to 25 ° C. and the shelf is heated. After further drying for 72 hours in this state, a FPOX lyophilized formulation is obtained.

  In the present invention, the chelating reagent that coexists to improve the stability of the FPOX lyophilized preparation is a compound that coordinates to a metal ion to give a chelating compound, and any compound that has such an action can be used. Even compounds can be used. For example, ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”), trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (hereinafter referred to as “CyDTA”), dimethylenetriamine-N, N, N ′ , N ″, N ″ -pentaacetic acid (DTPA), glycol ether diamine tetraacetic acid (EGDTA), 1,6-hexenediaminediamine-N, N, N ′, N′-tetraacetic acid (HDTA), triethylenetetramine hexaacetic acid (HDTA) TTHA), polyaminecarboxylic acid-based chelating reagents such as aspartic acid, and oxycarboxylic acid-based chelating reagents such as citric acid, malic acid, and isocitric acid. Preferably, polyaminecarboxylic acid-based chelating reagents include EDTA and CyDTA, and oxycarboxylic acid-based chelating reagents include citric acid and malic acid. In the present invention, the presence, type and number of salts coordinated with the chelating reagent are not limited at all, but for example, free acid compounds and sodium salt compounds are preferably used. The chelating reagents can be used alone or in combination.

  In the present invention, any sugar alcohol can be used as the sugar alcohol to be coexisted in order to improve the stability of the FPOX lyophilized preparation. Examples include trehalose, xylitol, sorbitol, inositol, mannitol, arabitol, dulcitol, erythritol and the like. In the present invention, although not limited to the above sugar alcohols, for example, trehalose is preferably used. The sugar alcohols can be used alone or in combination.

  In the present invention, any amino acid can be used as the amino acid to be coexisted in order to improve the stability of the FPOX lyophilized preparation. For example, sodium glutamate, glycine, lysine hydrochloride, asparagine, alanine, methionine, threonine and the like can be mentioned. In the present invention, the amino acids are not limited at all, but, for example, sodium glutamate is preferably used. The amino acids can be used alone or in combination.

  In the present invention, any metal salt can be used as the metal salt to be coexisted in order to improve the stability of the FPOX lyophilized preparation. Examples thereof include sodium chloride, potassium chloride, manganese chloride, magnesium chloride, ferrous chloride, calcium chloride, barium chloride, copper chloride and the like. In the present invention, although not limited to the above metal salts, for example, calcium chloride is preferably used. The above metal salts can be used alone or in combination.

  In the stabilization method of the present invention, the FPOX and the reagents described above are allowed to coexist. At this time, if necessary, other reagents may coexist. Usually, solid powders such as FPOX and reagents, or a solution thereof can be mixed together as appropriate.

  The concentration of FPOX used in the stabilization method of the present invention is not particularly limited, but is preferably 0.05 to 1000 U / ml, more preferably 0.10 to 400 U / ml. The enzyme titer here is defined as 1 U for the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using fructosyl valyl histidine as a substrate. The chelating reagent, sugar alcohols, amino acids, and metal salts may be prepared at any concentration as long as the FPOX stabilizing effect is exhibited and there is no inconvenience in handling the reagent containing the enzyme. be able to. For example, a preferable concentration of the chelating reagent is 5 mM or more, more preferably 10 to 200 mM. A preferred concentration of sugar alcohols is 5 mM or more, more preferably 20 to 500 mM. A preferred concentration of amino acids is 25 mM or more, more preferably 50 to 500 mM. A preferable concentration of the metal salt is 0.2 mM or more, more preferably 2 to 50 mM.

  In general, since the stability of an enzyme is greatly affected by the pH during storage, it is preferable to simultaneously use various buffers in a stable pH range as other reagents. The type of buffer solution used in the present invention, its concentration, and pH are not particularly limited. For example, the buffer solution has a buffer capacity between pH 6 and 10, and is set to a concentration that maintains a necessary and sufficient buffer capacity. It is desirable that Examples of such buffers include general-purpose tris buffers and phosphate buffers, good buffers such as BES, HEPES, TES, bicine, and tricine, and amino acid buffers such as glycine-NaOH. Boric acid buffer solution, Bis-Tris propane buffer solution, imidazole buffer solution and the like can also be used. About the density | concentration of a buffer solution, Preferably it is 5-500 mM, More preferably, it is 10-100 mM. In the present invention, Tris buffer or phosphate buffer is preferably used. When adding the chelating reagent, sugar alcohols, amino acids, and metal salts of the present invention to the buffer, add them directly or add, for example, their aqueous solution adjusted to pH 5-9, preferably 6-8. do it. When the pH deviates from the target range by adding the above-mentioned reagents, the pH is adjusted to fall within the target range by adding, for example, sodium hydroxide, potassium hydroxide, or aqueous ammonia. Is preferred.

  Further, as other reagents, for example, various inorganic salts such as sodium chloride, potassium chloride and sodium azide, polysaccharides such as dextran, bovine serum albumin (BSA), glycerol, surfactants Further, chemotherapeutic agents such as antibiotics and sulfa drugs may coexist. These reagents may be added to the buffer in advance.

  The stability is evaluated by storing the FPOX lyophilized preparation under various conditions in accordance with the storage conditions, transport conditions and measurement conditions of the enzyme actually used, and measuring the changes over time. In general, an abuse test is usually used to evaluate in a short time. For example, a method in which the FPOX freeze-dried preparation is kept warm at a constant high temperature and the residual activity is measured over time can be mentioned.

  In this way, the FPOX lyophilized preparation can be stabilized by allowing a chelating reagent, sugar alcohols, amino acids, and metal salts to coexist. Using this stabilization method, a stabilized FPOX lyophilized formulation containing FPOX and a chelating reagent, sugar alcohols, amino acids, and metal salts can be produced. For example, a pH 6-10 buffer solution containing sugar alcohol at a concentration of 5% or more is prepared, and FPOX (manufactured by Kikkoman) is added to this buffer solution so that the concentration is 0.1-400 U. Furthermore, since it is known that a freeze-dried preparation of an enzyme can generally be stabilized by coexisting a substrate, a fructosyl peptide may be added to 1 to 200 mM as necessary. This mixed solution is stirred, and the FPOX freeze-dried preparation can be produced by the method for producing a freeze-dried preparation. The FPOX lyophilized preparation of the present invention thus obtained is significantly stabilized as compared with the preparation produced by the conventional method, and is stabilized in a simple manner and at a low cost by the method of the present invention. A prepared FPOX lyophilized formulation can be produced.

Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples. However, the technical scope of the present invention is not limited by these examples.
<Experimental example 1>

(FPOX activity measurement method and stability test)
A method for measuring the amount of hydrogen peroxide will be described. Hereinafter, fructosyl valyl histidine is used as a substrate for measurement of the activity of the oxidase of the present invention unless otherwise specified. The enzyme titer was defined as 1 U for the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using fructosyl valyl histidine as a substrate.
A. Preparation of Reagent (1) Reagent 1: POD-4-aminoantipyrine solution 1.0 kU peroxidase (manufactured by Kikkoman), 100 mg of 4-aminoantipyrine (manufactured by Tokyo Chemical Industry), 0.1 M potassium phosphate buffer Dissolve in (pH 8.0) and make up to 1 L.
(2) Reagent 2: 2,4-dichlorophenol sulfate solution 25 ml of a commercially available 2% solution (manufactured by Nacalai) is dissolved in ion-exchanged water, and the volume is adjusted to 100 ml.
(3) Reagent 3: Substrate solution (150 mM; final concentration 5 mM)
Dissolve 624 mg of fructosyl valyl histidine in ion exchange water to a constant volume of 10 ml. Fructosylvaline histidine was prepared by the method described in JP-A No. 2001-95598.
B. Measurement method 2.7 ml of reagent 1, 100 μl of reagent 2, and 100 μl of FPOX enzyme solution (manufactured by Kikkoman) are mixed and preheated at 30 ° C. for 5 minutes. Thereafter, 100 μl of reagent 3 is added and mixed well, and then the absorbance at 510 nm is measured with a spectrophotometer (U-2000A, manufactured by Hitachi, Ltd.). The measured value is the change in absorbance per minute from 1 minute to 3 minutes after 510 nm. The control solution was the same as described above except that 100 μl of ion exchange water was added instead of 100 μl of reagent 3. A hydrogen peroxide standard solution prepared in advance was used in place of the reagent 3 and ion-exchanged water in place of the enzyme solution, and a graph was prepared in which the relationship with the amount of generated dye was examined. Using this graph, the micromol of hydrogen peroxide produced per minute at 30 ° C. was calculated, and this value was used as the activity unit in the enzyme solution. For dilution of the FPOX enzyme solution for activity measurement, a 10 mM phosphate buffer solution (pH 8.0) containing 0.15% (w / v) BSA was used. The stability test of the FPOX lyophilized formulation was performed by comparing the residual activity after the abuse test. The residual activity of the enzyme solution after the abuse test was expressed as a relative amount (%) when the change in absorbance at the time of preparing the enzyme solution was taken as 100%.

(Coexistence effect of EDTA · 2Na, a chelating reagent)
First, as a test for adding no stabilizer (control), 337 U of FPOX-CE (Kikkoman) was added to 1 ml of 20 mM Tris-HCl buffer (pH 7.5) and lyophilized. This freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activity after the abuse was measured. As a result, as shown in Table 1, entry 1, it was found that the activity after abuse decreased to 48% as compared with the enzyme activity before abuse.

  Next, a 20 mM Tris-HCl buffer solution (pH 7.5) containing 5 mM EDTA · 2Na was prepared, and 337 U of FPOX-CE (manufactured by Kikkoman Corp.) was added to 1 ml of this buffer solution and freeze-dried. The freeze-dried powder was similarly abused at 37 ° C. for 2 weeks, and the enzyme activity after the abuse was measured. As a result, as shown in Table 1, entry 2, the activity of 77% was maintained as compared with the enzyme activity before the abuse. That is, it was found that the storage stability of the FPOX-CE lyophilized preparation was improved by adding 5 mM EDTA · 2Na (chelating reagent) to FPOX-CE and freeze-drying.

(Coexistence of oxycarboxylic acid chelating reagents)
A 20 mM Tris-HCl buffer solution (pH 7.5) containing 17 mM sodium citrate or 37 mM malic acid, which is an oxycarboxylic acid chelating reagent, was prepared. To 1 ml of this buffer solution, 337 U of FPOX-CE (Kikkoman) was added and lyophilized. This freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activity before and after the abuse was compared to evaluate the effect of stabilizing the chelating reagent. As a result, as shown in Table 1, entries 3 and 4, by adding any oxycarboxylic acid chelating reagent to FPOX-CE and freeze-drying, the storage stability of the FPOX-CE freeze-dried preparation can be improved. all right.

(Coexistence effect of trehalose, a sugar alcohol)
20 mM Tris-HCl buffer (pH 7.5) containing trehalose at concentrations of 26, 53, 132, and 264 mM were prepared, respectively. To 1 ml of this buffer solution, 337 U of FPOX-CE (Kikkoman) was added and lyophilized. This freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activities before and after the abuse were compared to evaluate the effect of stabilizing sugar alcohols. The results are shown in Table 1, entries 8-11. The addition of 26 mM trehalose had a residual activity rate of 72% (entry 8), but the addition of 132 mM or higher trehalose resulted in a residual activity of 100% (entries 10, 11). That is, it was found that the storage stability of the FPOX-CE lyophilized preparation was significantly improved by adding trehalose with a high concentration of 132 mM or more.

(Coexistence effect of sodium L-glutamate, an amino acid)
A 20 mM Tris-HCl buffer solution (pH 7.5) containing 107 mM sodium L-glutamate was prepared. 337 U of FPOX-CE (manufactured by Kikkoman) was added to 1 ml of this buffer solution and freeze-dried. The freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activity before and after the abuse was compared to evaluate the effect of stabilizing amino acids. As a result, as shown in Table 1, entry 7, it was found that the activity of 98% was maintained as compared with the enzyme activity before abuse. That is, it was found that the storage stability of the FPOX-CE lyophilized preparation was significantly improved by adding sodium L-glutamate and freeze-drying.

(Coexistence effect of calcium chloride, one of the metal salts)
A 20 mM Tris-HCl buffer solution (pH 7.5) containing 10 mM calcium chloride was prepared. To 1 ml of this buffer solution, 337 U of FPOX-CE (Kikkoman) was added and lyophilized. The freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activity before and after the abuse was compared to evaluate the effect of stabilizing metal salts. As a result, as shown in Table 1, entry 6, it was found that 57% of the activity was maintained as compared with the enzyme activity before abuse. That is, it was found that the storage stability of the FPOX-CE lyophilized preparation was slightly improved by adding calcium chloride and lyophilization.

(Coexistence effect of EDTA · 2Na and sodium L-glutamate)
A 20 mM Tris-HCl buffer solution (pH 7.5) containing 5 mM EDTA · 2Na and 107 mM sodium L-glutamate was prepared. To 1 ml of this buffer solution, 337 U of FPOX-CE (Kikkoman) was added and lyophilized. The freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activities before and after the abuse were compared to evaluate the effect of stabilizing the coexistence of the chelating reagent and amino acids. As a result, as shown in Table 1, entry 12, it was found that the activity of 100% was maintained as compared with the enzyme activity before abuse. That is, it was found that by adding EDTA · 2Na and sodium L-glutamate and freeze-drying, the storage stability of the FPOX-CE freeze-dried preparation was dramatically improved.

(Coexistence effect of various stabilizers for FPOX-EE)
20 mM Tris-HCl buffer (pH 7.5) containing 53, 132, and 264 mM trehalose and 20 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride were prepared, respectively, and 264 mM trehalose and A 20 mM Tris-HCl buffer solution (pH 7.5) containing 2 mM calcium chloride was prepared. After adding 100 U of FPOX-EE (manufactured by Kikkoman) to 1 ml of these buffers, the solution was freeze-dried. The freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activities before and after the abuse were compared to evaluate the effects of various stabilizers. The results are shown in Table 2 and entries 2-6. On the other hand, 100 U of FPOX-CE (manufactured by Kikkoman Corp.) was added to 1 ml of 20 mM Tris-hydrochloric acid buffer (pH 7.5) and freeze-dried as a test with no stabilizer added (control). This freeze-dried powder was abused at 37 ° C. for 2 weeks, and the enzyme activity after the abuse was measured. The results are shown in Table 2, entry1.

  When the stabilizer was not added (entry 1), the same abuse test as described above was performed. As a result, the residual activity rate of FPOX-EE (manufactured by Kikkoman Corporation) was only 24%. However, when 2 mM calcium chloride (entry 2) was added, the enzyme activity after abuse improved about twice as much as entry 1, and when trehalose was added 132 mM or more (entries 4, 5), it showed 80% or more residual activity, and FPOX- It was found that the EE lyophilized formulation was stabilized. Further, when these stabilizers were combined (entry 6), the residual activity was 96%, indicating a better residual activity rate. That is, it was found that the storage stability of the FPOX-EE preparation freeze-dried was dramatically improved by simultaneously adding trehalose and calcium chloride and freeze-drying.

According to the present invention, by using a reagent other than sugar as a stabilizer for lyophilizing FPOX, an FPOX lyophilized preparation that does not cause an Amadori reaction in the reaction system at the time of glycated protein measurement and does not increase the background. And can be easily used in a measurement kit as a diagnostic enzyme for diabetes. Furthermore, it becomes possible to keep the enzyme stable for a long period of time during storage and transportation, and the development of a clinical diagnostic kit excellent in storage stability together with the present invention excellent in stability.

Claims (3)

A method for stabilizing a fructosyl peptide oxidase lyophilized preparation, characterized in that ethylenediaminetetraacetic acid and sodium glutamate coexist with fructosyl peptide oxidase. A method for stabilizing a fructosyl peptide oxidase lyophilized preparation, characterized in that calcium chloride and trehalose coexist with fructosyl peptide oxidase. A fructosyl peptide oxidase lyophilized preparation obtained by the method according to claim 1 or 2 .