A stable calcitonin calibrator and preparation method thereof

Abstract

The present invention provides a stable calcitonin calibrator and a preparation method thereof, belonging to the technical field of calibrator preparation. The calibrator comprises the following components: a buffer, a salt substance, a sugar substance, a polyol substance, a preservative, a protein substance, a composite protective agent and calcitonin; the composite protective agent is obtained by mixing sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mass ratio of 2:1:1. The calcitonin calibrator of the present invention has a validity period of up to 36 months, can be stable for 1 year under the condition of ‑20°C after re-dissolution, can be stable for 3 months under the condition of 2‑8°C after re-dissolution, and can be stable for 1 month under the condition of room temperature after re-dissolution, providing a stable and good-performance calibrator for calcitonin diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the technical field of calibrator preparation, and particularly relates to a stable calcitonin calibrator and a preparation method thereof. Background Art

[0002] Calcitonin (CT) is a single-chain polypeptide hormone composed of 32 amino acids with a molecular weight of 3418 Da. It is mainly secreted by parathyroid C cells of the thyroid gland. It has corresponding receptors in bones and kidneys. It has the function of regulating calcium and phosphorus metabolism and is an inhibitor of bone resorption, preventing bone loss during calcium stress (such as pregnancy, lactation and growth). Calcitonin is mainly used clinically for auxiliary diagnosis of thyroid and parathyroid function, including related examinations such as hyperparathyroidism, hypergastrinemia, and renal failure. Therefore, monitoring the concentration level of calcitonin in the human body is of great significance for the accurate and highly specific auxiliary diagnosis of parathyroid function diseases. As the core component calibrator of the reagent, its performance stability determines whether the reagent test results are reliable and accurate.

[0003] Sodium caseinate is a commonly used food additive. Due to its good stabilizing effect, it can increase the stability of antigens in the matrix and extend the shelf life of calibrants. However, sodium caseinate has the risk of coagulation when it is in solution for a long time, so it is considered to be used in calibrants of freeze-dried dosage forms. Sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose are widely used in the pharmaceutical and food industries. They have the functions of dispersion, disintegration and suspension. Therefore, they can effectively prevent protein aggregation and avoid coagulation, so that the protein is uniform and stable in the matrix, thereby promoting the efficacy of protein in protecting antigens; and because it can be used as a disintegrant, it can speed up the reconstitution of freeze-dried powder, shorten the reconstitution time, and save users time.

[0004] The calibrators currently available on the market not only have a short stability period, but also a limited storage period at -20°C after reconstitution, and are unstable at room temperature and 2-8°C after reconstitution, which causes inconvenience and waste to the use of calibrators. At the same time, their own instability will bring uncertain deviations to clinical tests. Therefore, it is very practical to develop a calibrator that is long-lasting and stable, and can extend the storage period to varying degrees at -20°C, 2-8°C and room temperature after reconstitution. Summary of the invention

[0005] The purpose of the present invention is to solve the problems that the existing calibrators have a short stability period, a limited storage period at -20°C after reconstitution, and are unstable at room temperature and 2-8°C after reconstitution, and to provide a stable calcitonin calibrator and a preparation method thereof.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention first provides a stable calcitonin calibrator, comprising the following components:

[0008] Buffers, salts, sugars, polyols, preservatives, proteins, compound protective agents and calcitonin;

[0009] The composite protective agent is obtained by mixing sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mass ratio of 2:1:1.

[0010] Preferably, the stable calcitonin calibrator comprises the following components:

[0011]

[0012] Preferably, the pH of the calcitonin calibrator is 7.5-8.0.

[0013] Preferably, the buffer is one or a combination of PB, HEPES, MOPS, sodium barbital-hydrochloric acid buffer, Tris-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer and potassium dihydrogen phosphate-sodium hydroxide buffer.

[0014] Preferably, the salt substance is one or both of sodium chloride and potassium chloride.

[0015] Preferably, the carbohydrate substance is one or a combination of trehalose, sucrose, maltose, lactose, mannose, glucan and fructan.

[0016] Preferably, the polyol substance is mannitol or sorbitol.

[0017] Preferably, the preservative is sodium azide or Proclin300.

[0018] Preferably, the protein substance is one or more of bovine serum albumin, human serum albumin and ovalbumin.

[0019] The present invention also provides a method for preparing a stable calcitonin calibrator, comprising:

[0020] Separately take purified water, add buffer, salt substances, sugar substances, polyol substances, preservatives, protein substances, and composite protective agents in order to dissolve, adjust the pH to 7.5-8.0, filter with a filter membrane, add calcitonin, and finally freeze-dry the prepared liquid calibrant to obtain a lyophilized product.

[0021] Beneficial effects of the present invention

[0022] The present invention provides a stable calcitonin calibrator and a preparation method thereof. The calcitonin calibrator, by using a composite protective agent composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose, improves the stability of the calcitonin calibrator: the validity period is up to 36 months, and it can be stable for 1 year at -20°C after re-dissolution, it can be stable for 3 months at 2-8°C after re-dissolution, and it can be stable for 1 month at room temperature after re-dissolution. The problem that the stability difference period of the calcitonin calibrator is short, the storage period at -20°C after re-dissolution is limited, and it is unstable at room temperature and 2-8°C after re-dissolution is solved. The calcitonin calibrator provided by the present invention not only has a long validity period, but also is easy to store after re-dissolution, and has high practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the real-time stability curve of the calcitonin calibrator.

[0024] Figure 2 This is the stability curve of the calcitonin calibrator at -20°C after reconstitution.

[0025] Figure 3 This is the stability curve of the calcitonin calibrator at 2-8°C after reconstitution.

[0026] Figure 4 This is the stability curve of the calcitonin calibrator at room temperature after reconstitution. DETAILED DESCRIPTION

[0027] The present invention first provides a stable calcitonin calibrator, comprising the following components:

[0028] Buffers, salts, sugars, polyols, preservatives, proteins, compound protective agents and calcitonin;

[0029] The composite protective agent is obtained by mixing sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mass ratio of 2:1:1.

[0030] It is further preferred that the calcitonin calibrator consists of the following components:

[0031]

[0032]

[0033] The pH of the calcitonin calibrator is 7.5-8.0.

[0034] According to the present invention, the buffer is preferably one or a combination of PB, HEPES, MOPS, sodium barbital-hydrochloric acid buffer, Tris-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer and potassium dihydrogen phosphate-sodium hydroxide buffer; the salt substance is preferably one or both of sodium chloride and potassium chloride; the sugar substance is preferably one or a combination of trehalose, sucrose, maltose, lactose, mannose, dextran and fructan; the polyol substance is preferably mannitol or sorbitol; the preservative is sodium azide or Proclin300; the protein substance is preferably one or more of bovine serum albumin, human serum albumin, ovalbumin mixed with sodium caseinate in any proportion.

[0035] The present invention also provides a method for preparing a stable calcitonin calibrator, comprising the following steps: taking 1L of purified water respectively, and then adding thereto a buffer solution, a salt substance, a sugar substance, a polyol substance, a preservative, a protein substance, and a composite protective agent of the concentration, adding and dissolving them in sequence, preferably adjusting the pH to 7.5-8.0 (25±1°C) with hydrochloric acid or a 0.1% sodium hydroxide solution, filtering once with a 0.22μm filter membrane, and then calculating the amount of calcitonin added according to the concentration, and finally freeze-drying the prepared liquid calibrator to obtain a freeze-dried product.

[0036] The technical solution of the present invention is described clearly and in detail below in conjunction with specific embodiments.

[0037] The equipment used in the following examples and comparative examples includes a 100,000-level electronic balance, a pH meter and a matching calibration buffer solution, a 1000 mL measuring cylinder and a special filter, and a vacuum freeze dryer.

[0038] Comparative Example 1

[0039] The calcitonin calibrator provided by the present invention has the following components and concentrations:

[0040]

[0041]

[0042] According to the above concentration ratio: add the whole batch of purified water to a clean preparation container, and then add the whole batch of buffer, salt substances, sugar substances, polyol substances, preservatives, and bovine serum albumin in order. After adding and dissolving in order, adjust the pH to 7.8 (25±1°C) with hydrochloric acid or 0.1% sodium hydroxide solution, and filter once with a 0.22μm filter membrane. Then add calcitonin according to the concentration calculation, so that the concentration of the calibration product is 5pg / mL for the low value and 500pg / mL for the high value. According to the weighing method of 1g±0.005g / bottle, the packaged liquid calibration product is packaged and freeze-dried to obtain a freeze-dried product. Add 1g±0.005g of purified water to the prepared freeze-dried calibration product, let it stand at room temperature for re-dissolution, and then mix them separately for use.

[0043] Comparative Example 2

[0044] The difference from Comparative Example 1 is that 0.2% sodium caseinate, 0.2% sodium hydroxypropyl methylcellulose and 0.2% sodium carboxymethyl cellulose are added respectively.

[0045] The calibration product prepared in the above comparative proportion is processed as follows:

[0046] The calibrators to be tested were stored at 2-8°C and 37°C for 14 days, and the effects of different protective agents on the stability of the calcitonin calibrators were tested using a calcitonin assay kit (chemiluminescence immunoassay) and a CM-180 fully automatic chemiluminescence immunoassay analyzer. The smaller the decay rate, the better (Table 1);

[0047] Table 1 14-day high temperature accelerated stability of calcitonin calibrators with different protective agents

[0048]

[0049]

[0050] Note: Calibrator concentration unit: pg / mL

[0051] It can be seen from Table 1 that sodium caseinate has a significant protective effect on the calcitonin calibrator, while sodium hydroxypropyl methylcellulose and sodium carboxymethylcellulose have no significant protective effect on the calcitonin calibrator.

[0052] Comparative Example 3

[0053] The calcitonin calibrator provided by the present invention has the following components and concentrations:

[0054]

[0055] According to the above concentration ratio: add the whole batch of purified water to a clean preparation container, and then add the whole batch of buffer, salt substances, sugar substances, polyol substances, preservatives, bovine serum albumin, sodium caseinate in order, add them in order and dissolve them, adjust the pH to 7.8 (25±1℃) with hydrochloric acid or 0.1% sodium hydroxide solution, and filter once with a 0.22μm filter membrane. Then add calcitonin according to the concentration calculation, so that the concentration of the calibration product is 5pg / mL for the low value and 500pg / mL for the high value. According to the weighing method, 1g±0.005g / bottle is packaged, and finally the packaged liquid calibration product is freeze-dried to obtain a freeze-dried product. Add 1g±0.005g of purified water to the prepared freeze-dried calibration product, let it stand at room temperature for re-dissolution, and then mix them separately for use.

[0056] Example 1

[0057] The difference from Comparative Example 3 (Group A) is that 0.2% sodium caseinate is replaced with the following combination: Group B is a 0.2% combination, i.e. sodium caseinate and sodium hydroxypropyl methylcellulose are mixed in a mass ratio of 1:1; Group C is a 0.2% combination, i.e. sodium caseinate and sodium carboxymethylcellulose are mixed in a mass ratio of 1:1; Group D is a 0.2% combination, i.e. sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethylcellulose are mixed in a mass ratio of 2:1:1.

[0058] (1) Take the calibrant to be tested and accurately reconstitute it by adding 1 g ± 0.005 g of purified water to each bottle. Use a timer to accurately record the time required for the lyophilized powder to completely dissolve when left to stand. The faster the dissolution rate, the more suitable it is for clinical use (Table 2);

[0059] Table 2 Redissolution time of calcitonin calibrators with different protective agent combinations

[0060]

[0061] (2) Take the calibrant to be tested, add 1g±0.005g purified water to each bottle and accurately reconstitute it, then place it at 2-8℃, record the appearance changes at different cycles and verify the decay rate. The appearance is light yellow liquid with no obvious insoluble matter, which is qualified, and the decay rate is not more than 5% is qualified (Table 3, Table 4)

[0062] Table 3 Appearance record of calcitonin calibrators with different protective agent combinations after reconstitution at 2-8℃

[0063]

[0064]

[0065] Table 4 Attenuation rate of calcitonin calibrators after reconstitution with different protective agent combinations at 2-8℃

[0066]

[0067] Note: The concentration of the calibrator is in pg / mL

[0068] (3) Take the calibrant to be tested, add 1g±0.005g purified water to each bottle and accurately reconstitute it. Place it at room temperature, record the appearance changes at different cycles and verify the decay rate. The appearance is light yellow liquid, no obvious insoluble matter, and the decay rate is not more than 5% to be qualified (Table 5, Table 6)

[0069] Table 5 Appearance record of calcitonin calibrators with different protective agent combinations after reconstitution at room temperature

[0070]

[0071]

[0072] Table 6 Attenuation rate of calcitonin calibrators after reconstitution with different protective agent combinations at room temperature

[0073]

[0074] Note: The concentration of the calibrator is in pg / mL

[0075] As shown in Table 2, both sodium hydroxypropyl methylcellulose and sodium carboxymethylcellulose can accelerate the dissolution rate of freeze-dried powder, and the shorter the dissolution time, the more convenient it is for clinical use when both are increased at the same time. As shown in Tables 3-6, both sodium hydroxypropyl methylcellulose and sodium carboxymethylcellulose can promote the protective effect of sodium caseinate on the calcitonin calibrator, and the protective effect of the two on the calcitonin calibrator is significantly improved after they are combined with sodium caseinate. In summary, the composite protective agent composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethylcellulose in a mass ratio of 2:1:1 has the most obvious protective effect on the calcitonin calibrator, and considering that the dissolution will become more and more difficult as the amount of the three added increases, the concentration range of the composite protective agent is 0.2%-2%.

[0076] After determining that the composite protective agent is sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mass ratio of 2:1:1, the calcitonin calibrator with a usage of 0.2% of the composite protective agent is selected, and the calcitonin assay kit (chemiluminescent immunoassay) is used. The CM-180 fully automatic chemiluminescent immunoassay is used for verification, and the changes in the test results of the calcitonin calibrator are recorded and analyzed, and a calcitonin calibrator stability tracking curve is formed. Figure 1 As shown: the concentration of the calibrator is measured every 6 months until 36 months, forming a real-time stability curve; Figure 2As shown in Table 7: Verification of homogeneity indicators.

[0077] Table 7 Calcitonin Calibrator Homogeneity Test Data

[0078]

[0079] Note: Calibrator concentration unit: pg / mL

[0080] In summary, the real-time stability of the calcitonin calibrator of the present invention (under the condition of 2-8°C) is at least 36 months, after reconstitution, it is stable at least for 1 year under the condition of -20°C, and after reconstitution, it is stable at least for 3 months under the condition of 2-8°C. Figure 3 As shown, it is stable at room temperature for at least 1 month. Figure 4 Therefore, it is determined that the composite protective agent composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a ratio of 2:1:1 can improve the stability of the calcitonin calibrator, and has stable and uniform performance, and can be used as a supporting reagent as a reagent supporting calibrator.

[0081] Example 2

[0082] The calcitonin calibrator provided by the present invention has the following components and concentrations:

[0083]

[0084] Note: The composite protective agent is composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose mixed in a mass ratio of 2:1:1.

[0085] According to the above concentration ratio: add the whole batch of purified water to a clean preparation container, and then add the whole batch of buffer, salt substances, sugar substances, polyol substances, preservatives, ovalbumin, and composite protective agents in order. After dissolving in order, adjust the pH to 7.8 (25±1°C) with hydrochloric acid or 0.1% sodium hydroxide solution, and filter once with a 0.22μm filter membrane. Then add calcitonin according to the concentration calculation, so that the concentration of the calibration product is 5pg / mL for the low value and 500pg / mL for the high value. According to the weighing method of 1g±0.005g / bottle, the packaged liquid calibration product is packaged and freeze-dried to obtain a freeze-dried product. Add 1g±0.005g of purified water to the prepared freeze-dried calibration product, let it stand at room temperature for re-dissolution, and then mix them separately for use.

[0086] Example 3

[0087] The calcitonin calibrator provided by the present invention has the following components and concentrations:

[0088]

[0089] Note: The composite protective agent is composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose mixed in a mass ratio of 2:1:1.

[0090] According to the above concentration ratio: add the whole batch of purified water to a clean preparation container, and then add the whole batch of buffer, salt substances, sugar substances, polyol substances, preservatives, human serum albumin, and composite protective agents in order. After dissolving in order, adjust the pH to 7.8 (25±1°C) with hydrochloric acid or 0.1% sodium hydroxide solution, and filter once with a 0.22μm filter membrane. Then add calcitonin according to the concentration calculation, so that the concentration of the calibration product is 5pg / mL for the low value and 500pg / mL for the high value. According to the weighing method of 1g±0.005g / bottle, the packaged liquid calibration product is packaged and freeze-dried to obtain a freeze-dried product. Add 1g±0.005g of purified water to the prepared freeze-dried calibration product, let it stand at room temperature for re-dissolution, and then mix them separately for use.

[0091] Example 4

[0092] The calcitonin calibrator provided by the present invention has the following components and concentrations:

[0093]

[0094]

[0095] Note: The composite protective agent is composed of sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose mixed in a mass ratio of 2:1:1.

[0096] According to the above concentration ratio: add the whole batch of purified water to a clean preparation container, and then add the whole batch of buffer, salt substances, sugar substances, polyol substances, preservatives, bovine serum albumin, and composite protective agents in order. After dissolving in order, adjust the pH to 7.8 (25±1°C) with hydrochloric acid or 0.1% sodium hydroxide solution, and filter once with a 0.22μm filter membrane. Then add calcitonin according to the concentration calculation, so that the concentration of the calibration product is 5pg / mL for the low value and 500pg / mL for the high value. According to the weighing method of 1g±0.005g / bottle, the liquid calibration product is packaged and freeze-dried to obtain a freeze-dried product. Add 1g±0.005g of purified water to the prepared freeze-dried calibration product, let it stand at room temperature for re-dissolution, and then mix them separately for use.

[0097] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A stable calcitonin calibrator, It is characterized in that Includes the following components: Buffer 5-20mmol / mL; Salt substances 80-150mmol / mL; Sugar substances 1-10%; Polyols 1-10%; Preservatives 0.05-0.2%; Protein substances 0.05-5%; Composite protective agent 0.2%-2%; Calcitonin 1-6pg / mL or 100-600pg / mL; The composite protective agent is obtained by mixing sodium caseinate, sodium hydroxypropyl methylcellulose and sodium carboxymethyl cellulose in a mass ratio of 2:1:

1.

2. A stable calcitonin calibrator according to claim 1, It is characterized in that The pH of the calcitonin calibrator is 7.5-8.

0.

3. A stable calcitonin calibrator according to claim 1, It is characterized in that The buffer is one or a combination of PB, HEPES, MOPS, sodium barbital-hydrochloric acid buffer, Tris-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer and potassium dihydrogen phosphate-sodium hydroxide buffer.

4. A stable calcitonin calibrator according to claim 1, It is characterized in that The salt substance is one or both of sodium chloride and potassium chloride.

5. A stable calcitonin calibrator according to claim 1, It is characterized in that The sugar substance is one or a combination of trehalose, sucrose, maltose, lactose, mannose, glucan and fructan.

6. A stable calcitonin calibrator according to claim 1, It is characterized in that The polyol substance is mannitol or sorbitol.

7. A stable calcitonin calibrator according to claim 1, It is characterized in that The preservative is sodium azide or Proclin300.

8. A stable calcitonin calibrator according to claim 1, It is characterized in that The protein substance is one or more of bovine serum albumin, human serum albumin and ovalbumin.

9. A method for preparing a stable calcitonin calibrator according to claim 1, It is characterized in that include: Separately take purified water, add buffer, salt substances, sugar substances, polyol substances, preservatives, protein substances, and composite protective agents in order to dissolve, adjust the pH to 7.5-8.0, filter with a filter membrane, add calcitonin, and finally freeze-dry the prepared liquid calibrant to obtain a lyophilized product.

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