JP5759211B2 - Freeze drying method - Google Patents

Description

  The present invention relates to a freeze-drying method for maintaining long-term stability without reducing the activity of a troponin complex or free troponin T.

Immunoassays are often used to detect proteins in serum or urine samples for medical diagnostic purposes because of their high specificity and sensitivity. Such immunoassays require calibrators used as reference standards for quantifying patient samples in addition to measurement reagents. Usually, the antigen contained in this calibrator is dilute, and depending on the antigen species, it may be remarkably inactivated in solution. In particular, antigen solutions such as hormones, enzymes, and insoluble proteins are severely inactivated and are often difficult to use as aqueous calibration materials. In such cases, store in a form that has been dehydrated by methods such as freeze-drying, and add and dissolve water at the time of use, or add proteins such as bovine serum albumin or animal serum in an aqueous solution. Measures have been taken to ensure stability (see, for example, Non-Patent Documents 1 and 2).
However, proteins that are involved in muscle contraction, such as troponin, are part of the insoluble protein complex, and purified troponin complex or free troponin T is so unstable that it changes its apparent conformation. Alternatively, the activity is remarkably reduced due to adhesion to the surface of the container, and there is a problem that it is difficult to ensure stability with the conventional method.

Jun Sasaki, immunochemical identification method (3rd edition), Tokyo Chemical Doujin, 1993 Yuji Ishikawa, ultrasensitive enzyme immunoassay, Academic Publishing Center, 1993

  An object of the present invention is to provide a lyophilization method of an aqueous solution containing a troponin complex or free troponin T excellent in storage stability of the troponin complex or free troponin T.

The inventor of the present invention has arrived at the present invention as a result of intensive investigations to achieve the above object. That is, the present invention is a lyophilization method of an aqueous solution (A) containing a troponin complex or free troponin T, wherein the (A) is heated at 30 to 50 ° C. for 72 to 240 hours, and then sulfated. The lyophilization method is characterized by lyophilization in the presence of a type anionic surfactant (B2) .

  By the freeze-drying method of the present invention, the storage stability of a troponin complex or free troponin T whose activity is likely to be reduced can be improved and the activity can be maintained for a long time.

  The troponin complex in the present invention plays a role in calcium-dependent regulation of muscle contraction and relaxation. Free troponin T is one of the three subunits constituting the troponin complex, and plays an important role in regulating muscle contraction by adjusting the binding between tropomyosin and actin. Since these components do not appear in the blood when there is no muscle damage, it is considered that there was muscle damage when detected in the blood. Therefore, the measurement of troponin is specific to the myocardium and is a powerful index for diagnosis of myocardial infarction.

Troponin complex and free troponin T are of human or animal origin, more particularly of cardiac origin.
Troponin complexes are obtained from extracts of heart milling preparations, and free troponin T is obtained from purified troponin complexes. In addition, a stable composition of troponin complex or free troponin T has been described in American Heart Journal, Clinical Investments, June 1987, Vol. 113, No. 6. “Cardiac-specific troponin-I radioimmunoassay in the diagnosis of acute myocardial infection” Manufactured from human or animal-derived hearts using the method disclosed on the page. It may be the one.
Examples of commercially available free troponin T include those manufactured by Bios Pacific or Fitzgerald.

The aqueous solution (A) containing the troponin complex or free troponin T contains the troponin complex or free troponin T and water.
Although it does not specifically limit as water, Deionized water is preferable from a viewpoint of purity.

  The content of the troponin complex or free troponin T in the aqueous solution (A) containing the troponin complex or free troponin T is 10 mg / g based on the weight of the aqueous solution (A) from the viewpoint of storage stability. The following is preferable, and from the viewpoint of a concentration range necessary for quantifying troponin complex or free troponin T in blood, more preferably 10 pg / g to 1 μg / g, particularly preferably 10 pg / g to 100 ng / g, Most preferably, it is 10 pg / g-25 ng / g.

  The aqueous solution (A) containing the troponin complex or free troponin T preferably contains a protein for the purpose of preventing adsorption of the troponin complex or free troponin T to the container. Examples of the protein include bovine serum albumin and other proteins (human serum albumin, fetal bovine serum, horse serum, bovine gamma globulin, and the like).

  When the protein is contained, the content of the protein in the aqueous solution (A) is preferably 0.1 to 5% by weight based on the weight of the aqueous solution (A) from the viewpoint of the stability of the troponin complex or free troponin T. %, More preferably 0.3 to 3% by weight, particularly preferably 0.5 to 3% by weight.

The pH of the aqueous solution (A) containing the troponin complex or free troponin T is preferably 6.0 to 8.5, more preferably 6.5 to 8.0, particularly preferably from the viewpoint of storage stability. 6.5-7.5. When the pH is less than 6.0 or exceeds 8.5, the stability of the troponin complex or free troponin T tends to deteriorate.
The pH is a value at a measurement temperature of 25 ° C. measured based on JIS K0400-12-10: 2000.

  In order to adjust the pH of the aqueous solution (A) containing the troponin complex or free troponin T to the above preferred range, a known buffer solution such as a phosphate buffer solution or a Good buffer solution can be used. From the viewpoint of stability of the troponin complex or free troponin T, a phosphate buffer is preferable.

  The aqueous solution (A) containing the troponin complex or free troponin T can further contain a nonionic surfactant for the purpose of stabilizing the troponin complex or free troponin T.

  Nonionic surfactants include alkylene oxide (hereinafter abbreviated as AO) addition type nonionic surfactants [higher alcohol AO adduct, alkylphenol AO adduct, polypropylene glycol ethylene oxide adduct, polyethylene glycol propylene oxide addition. Products and fatty acid AO adducts] and polyhydric alcohol type nonionic surfactants.

  Examples of AO in the nonionic surfactant include ethylene oxide (hereinafter abbreviated as EO), propylene oxide (hereinafter abbreviated as PO), butylene oxide, and the like.

  Examples of the AO adduct of higher alcohols include EO 1-20 mol adducts of higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.), and 8 to 24 carbon atoms. And PO1-40 mol adducts of higher alcohols.

  Examples of the AO adduct of alkylphenol include an EO 1-40 mol adduct of C7-C36 alkylphenol (nonylphenol, cetylphenol, etc.), and a PO1-40 mol adduct of C7-C36 alkylphenol.

  Examples of the polypropylene glycol EO adduct include a pluronic-type surfactant, and an EO 2-40 mol adduct of polypropylene glycol having 10 to 100 propylene oxide repeating units.

  Examples of the polyethylene glycol PO adduct include a PO 2-20 mol adduct of polyethylene glycol having 10 to 100 ethylene oxide repeating units.

  Examples of fatty acid AO adducts include EO 1 to 25 mol adducts of higher fatty acids having 8 to 24 carbon atoms (such as lauric acid, stearic acid and oleic acid).

  Examples of the polyhydric alcohol type nonionic surfactant include fatty acids (8 to 24 carbon atoms) of 2 to 8 carbon polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan). EO and / or PO adducts of fatty acid (carbon number 8-24) ester of fatty acid (carbon number 8-24) ester and fatty acid (carbon number 10-18) alkanolamide (lauric acid) Monoethanolamide and lauric acid diethanolamide).

  Among these, from the viewpoint of the stability of the troponin complex or free troponin T, an AO adduct of a higher alcohol and a fatty acid (carbon number 8 to 24) ester of a divalent to octavalent polyhydric alcohol having 3 to 36 carbon atoms. EO and / or PO adducts are more preferred, and more preferred are EO and / or PO adducts of fatty acid (carbon number 8 to 24) esters of divalent to octavalent polyhydric alcohols having 3 to 36 carbon atoms, Particularly preferred is a sorbitan fatty acid (carbon number 8 to 24) ester EO 6 to 40 mol adduct. Specific examples of particularly preferable ones include TWEEN 80.

  When the nonionic surfactant is contained, the content is preferably 0.1 to 1% by weight from the viewpoint of the stability of the troponin complex or free troponin T, based on the weight of the aqueous solution (A). More preferably, it is 0.1 to 0.5 weight%, Most preferably, it is 0.1 to 0.3 weight%.

  The aqueous solution (A) containing the troponin complex or free troponin T is obtained by mixing the troponin complex or free troponin T, water, and if necessary, a buffer, a protein, a nonionic surfactant and other components. Easy to get. Other ingredients may include salt to provide a physiological serum environment.

  After the heat treatment of the aqueous solution (A) containing the troponin complex or free troponin T, the storage stability of the troponin complex or free troponin T is improved by adding an anionic surfactant (B) and freeze-drying. Can be improved.

The temperature of the heat treatment needs to be 30 to 50 ° C., and the heat treatment time needs to be 72 to 240 hours.
From the viewpoint of the stability of the troponin complex or free troponin T, the temperature of the heat treatment is preferably 30 to 45 ° C, more preferably 35 to 45 ° C, and particularly preferably 38 to 42 ° C.
The heat treatment time is preferably 96 to 220 hours, more preferably 120 to 200 hours, and particularly preferably 150 to 180 hours from the viewpoint of the stability of the troponin complex or free troponin T.
If the treatment temperature is less than 30 ° C, the stabilizing effect is small, and if the treatment temperature exceeds 50 ° C, the troponin complex or free troponin T is denatured. Similarly, when the treatment time is less than 72 hours, the stabilizing effect is small, and when the treatment time exceeds 240 hours, the troponin complex or free troponin T is denatured.

  The apparatus for heating is not particularly limited as long as it can be set to the above-described treatment temperature and time, and examples thereof include an incubator.

  Examples of the anionic surfactant (B) include a carboxylate type anionic surfactant (B1), a sulfate ester type anionic surfactant (B2), a sulfonate type anionic surfactant (B3), and a phosphate ester salt. Type anionic surfactant (B4) and the like.

  As the carboxylate anionic surfactant (B1), a saturated or unsaturated fatty acid having 8 to 22 carbon atoms (capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid Linoleic acid, ricinoleic acid, coconut oil, palm kernel oil, rice bran oil, and beef tallow), carboxymethylated salts of aliphatic alcohols having 8 to 16 carbon atoms, and EO1 of aliphatic alcohols having 8 to 16 carbon atoms. Examples thereof include a salt of a carboxymethylated product of 10 mol adduct.

Examples of the sulfate ester type anionic surfactant (B2) include higher alcohol sulfates having 8 to 18 carbon atoms, higher alkyl ether sulfates having 8 to 18 carbon atoms, sulfated oils (natural unsaturated fats and oils). Saturated waxes that have been neutralized by sulfation), sulfated fatty acid esters (sulfurized and neutralized lower alcohol esters of unsaturated fatty acids) and sulfated olefins (sulfated olefins having 12 to 18 carbon atoms) Neutralized).
Examples of the salt include sodium, potassium, ammonium, alkanolamine and the like.

  Examples of the sulfonate type anionic surfactant (B3) include alkylbenzene sulfonate, alkylnaphthalene sulfonate, sulfosuccinic acid diester type, α-olefin sulfonate, and Igepon T type.

  Examples of the phosphate ester type anionic surfactant (B4) include higher alcohol phosphate ester salts and higher alcohol EO adduct phosphate ester salts.

  Examples of the salt in (B1) to (B4) include sodium salt, potassium salt, ammonium salt, alkanolamine salt and the like.

Among these, from the viewpoint of stability of the troponin complex or free troponin T, sulfate ester type anionic surfactant (B2) is preferable, and more preferable is higher alcohol sulfate ester having 8 to 18 carbon atoms. Particularly preferred is lauryl alcohol sulfate.
Specific examples of particularly preferable ones include sodium lauryl sulfate.

  The content of the anionic surfactant (B) is preferably 0.1 to 0.5% by weight from the viewpoint of the stability of the troponin complex or free troponin T, based on the weight of the aqueous solution (A). Preferably it is 0.1 to 0.3 weight%, Most preferably, it is 0.15 to 0.25 weight%.

  It is preferable that the temperature at the time of mixing the aqueous solution (A) after heat processing and an anionic surfactant (B) is 15-30 degreeC. The mixing time of (A) and (B) is not particularly limited as long as (B) can be dissolved, but it is preferably 10 minutes to 2 hours.

  The lyophilization method is not particularly limited. For example, an aqueous solution obtained by adding an anionic surfactant (B) to the aqueous solution (A) after the heat treatment is weighed in a glass bottle and placed in a freeze dryer (manufactured by ULVAC). Thereafter, a method of freezing at a shelf temperature of −30 ° C. for 3 hours and drying under reduced pressure (0.05 mmHg) for 24 hours may be mentioned. These conditions of temperature, time, and degree of vacuum can be changed as appropriate.

  The freeze-drying method of the present invention can be used when performing immunoassays such as enzyme immunoassay, radioimmunoassay, and immunoturbidimetry in the field of clinical examination. In particular, the storage stability of the troponin complex or free troponin T is improved, and its activity can be maintained for a long time, so that it can be suitably used as a calibration substance.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.

<Example 1>
To 10 mL of deionized water, 8 mg of potassium dihydrogen phosphate, 50 mg of disodium hydrogen phosphate and 0.85 g of sodium chloride were added and stirred. Further, 0.1 g of bovine serum albumin and 0.03 g of nonionic surfactant (Tween 80) were added and stirred well. Thereafter, free troponin T was added to 10 ng / mL and stirred to obtain an aqueous solution (A-1). Thereafter, the aqueous solution (A-1) was heated in an incubator at 40 ° C. for 168 hours, 0.02 g of sodium lauryl sulfate as the anionic surfactant (B) was added, and the mixture was stirred at 20 ° C. for 30 minutes. Thus, an aqueous solution containing free troponin T was obtained. 1.05 mL of this aqueous solution was weighed into a glass bottle and placed in a freeze dryer (ULVAC). The shelf temperature of the freeze dryer was set to −30 ° C. and frozen for 3 hours, and then dried under reduced pressure (0.05 mmHg) for 24 hours.

<Example 2>
Lyophilization was performed in the same manner as in Example 1 except that the temperature of the heat treatment of the aqueous solution (A-1) was changed from 40 ° C to 50 ° C.

<Example 3>
Lyophilization was performed in the same manner as in Example 1 except that the temperature of the heat treatment of the aqueous solution (A-1) was changed from 40 ° C to 30 ° C.

<Example 4>
Lyophilization was performed in the same manner as in Example 1 except that the heat treatment time of the aqueous solution (A-1) was changed from 168 hours to 240 hours.

<Example 5>
Lyophilization was performed in the same manner as in Example 1 except that the time for the heat treatment of the aqueous solution (A-1) was changed from 168 hours to 72 hours.

<Example 6>
Lyophilization was performed in the same manner as in Example 1 except that the amount of sodium lauryl sulfate added was changed from 0.02 g to 0.05 g.

<Example 7>
Lyophilization was performed in the same manner as in Example 1 except that the amount of sodium lauryl sulfate added was changed from 0.02 g to 0.01 g.

<Example 8>
Lyophilization was performed in the same manner as in Example 1 except that sodium lauryl sulfate was changed to oleyl sulfate sodium salt.

<Example 9>
Lyophilization was performed in the same manner as in Example 1 except that bovine serum albumin and a nonionic surfactant (Tween 80) were not added.

<Comparative Example 1>
Lyophilization was performed in the same manner as in Example 1 except that the aqueous solution (A-1) was not subjected to heat treatment.

<Comparative Example 2>
Lyophilization was performed in the same manner as in Example 1 except that the temperature of the heat treatment of the aqueous solution (A-1) was changed from 40 ° C to 25 ° C.

<Comparative Example 3>
Lyophilization was performed in the same manner as in Example 1 except that the temperature of the heat treatment of the aqueous solution (A-1) was changed from 40 ° C to 55 ° C.

<Comparative Example 4>
Lyophilization was performed in the same manner as in Example 1 except that the time for the heat treatment of the aqueous solution (A-1) was changed from 168 hours to 300 hours.

<Comparative Example 5>
Lyophilization was performed in the same manner as in Example 1 except that the time for the heat treatment of the aqueous solution (A-1) was changed from 168 hours to 48 hours.

<Comparative Example 6>
Lyophilization was performed in the same manner as in Example 1 except that sodium lauryl sulfate was not used.

<Measurement of light emission>
After adding 1.0 mL of deionized water to the lyophilized products obtained in Examples 1 to 9 and Comparative Examples 1 to 6, stirring 10 times by pipetting and allowing to stand for 1 minute, then fully automatic chemiluminescence immunization The amount of luminescence (the amount of luminescence immediately after production) was measured with a measuring device [Sphere Light (registered trademark) 180, manufactured by Olympus Corporation]. The lyophilized product was stored in a refrigerator at 2 to 10 ° C. for 6 or 13 months, and the light emission amount (light emission amount after 6 months or light emission after 13 months) was measured in the same manner.
Table 1 shows the value of each light emission amount and the retention rate (%) of the light emission amount after 6 months and the light emission amount after 13 months based on the light emission amount immediately after preparation of the freeze-dried product.

  Since the lyophilization method of the present invention can stably store a troponin complex or free troponin T for a long period of time, it can be used in the field of clinical testing by enzyme immunoassay, radioimmunoassay, immunoturbidimetry, Useful when performing immunoassays. In particular, since it can prevent inactivation of the antigen in the solution containing the antigen and can be stably stored, it can be suitably used as a standard solution.

Claims (4)

A lyophilization method of an aqueous solution (A) containing a troponin complex or free troponin T, wherein the (A) is heat-treated at 30 to 50 ° C. for 72 to 240 hours, and then a sulfate ester type anionic surfactant A freeze-drying method characterized by freeze-drying in the presence of (B2) . The sulfuric acid ester salt type anionic surfactant (B2) is, freeze drying method according to claim 1 Symbol placement higher alcohol sulfates having 8 to 18 carbon atoms. The freeze-drying method according to claim 1 or 2 , wherein the aqueous solution (A) further contains bovine serum albumin. The lyophilization method according to any one of claims 1 to 3 , wherein the aqueous solution (A) further contains a nonionic surfactant.