A B-type natriuretic peptide stabilizer and its preparation method and application

Abstract

The present invention discloses a B-type natriuretic peptide stabilizer and its preparation method and application. The stabilizer includes the following components: 10-90 v / v% human serum matrix, 5-25g / L trehalose, 10-25g / L mannitol, 1-5mM N-acetylcysteine, 1.0-2.5g / L fish gelatin, 0.02-0.1 v / v% preservative, 0.02-0.1 v / v% 5-bromo-5-nitro-1,3-dioxane. The stabilizer of the present invention can improve the stability of B-type natriuretic peptide BNP in a serum-containing stabilizer, greatly improve the storage stability, effectively extend the shelf life in an independent quality control product for in vitro diagnosis, and improve its commercial value; at the same time, it improves the ability to resist qualitative changes during preparation and storage, and reduces the batch difference caused by the freeze-drying process.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a B-type natriuretic peptide stabilizer and a preparation method and application thereof. Background Art

[0002] B-type natriuretic peptide (BNP) and N-terminal B-type natriuretic peptide precursor (NT-proBNP) are biomarkers of cardiac function, and are also the preferred biomarkers for the diagnosis and differential diagnosis of heart failure, the severity of the disease and the evaluation of prognosis. With the continuous deepening of clinical research and the widespread application of new heart failure treatment drugs. BNP is a member of the natriuretic peptide family secreted by the heart. The main structure of BNP in human plasma is a 32-peptide with a specific ring structure, with a ring-shaped central structure containing 17 amino acid residues composed of two cysteines connected by an intramolecular disulfide bond. The activity of BNP is particularly dependent on its ring-shaped central structure. BNP is species-specific. The human BNP gene fragment is located at the distal end of the short arm of chromosome 1 and contains 3 exons and 2 introns. Its reverse transcribed deoxyribonucleic acid (cDNA) consists of 1900 nucleotides, and the messenger RNA (mRNA) of BNP consists of 900-1000 nucleotides, which are then explosively translated into pre-proBNP consisting of 134 amino acids. Under the action of proteases, this polypeptide is rapidly decomposed into a 26-amino acid signal peptide and proBNP containing a 108-amino acid peptide. Subsequently, proBNP is decomposed by type II transmembrane serine proteases into an inactive 76-amino acid peptide (the N-terminus of the brain natriuretic peptide precursor) and a 32-amino acid peptide BNP with endocrine activity. The pre-proBNP and pro-BNP formed after expression in the above process are unstable and decompose quickly. BNP is relatively stable and is completely released into the blood, with a high concentration and a long half-life, so BNP is often detected in clinical applications.

[0003] BNP is widely distributed in tissues such as the brain, spinal cord, heart and lungs, with the highest content in the heart. The highest content in the brain is in the medulla oblongata. The BNP content in the central nervous system is higher than that in ANP. The BNP content in the brain and spinal cord is about 13 times higher than that in ANP. BNP in the heart is mainly present in the left and right atria, with the content in the right atrium being more than 3 times that in the left atrium, and the BNP content in the ventricle being less than 1 / 20 of that in the atrium. The low BNP content in the ventricle is because the BNP precursor is not stored in the ventricle. Only when the ventricular wall tension increases will it rapidly stimulate high expression of the BNP gene, and a large amount of synthesized BNP will be secreted into the blood. In other words, very little BNP is stored in the ventricular muscle. A small amount of BNP is also contained in the atrial septum, atrioventricular valve, aorta, hepatic artery and pulmonary vein walls. BNP is a quantitative marker for heart failure. It is released by ventricular muscle cells after ventricular pressure increases and can reflect heart function. BNP has important pathophysiological significance. It can promote sodium excretion and urination, has a strong vasodilator effect, and can counteract the vasoconstrictor effect of the renin-angiotensin-aldosterone system (RAAS). Like ANP, it is a major endocrine system for the human body to resist excessive volume load and hypertension. Cardiac dysfunction can greatly activate the natriuretic peptide system, and increased ventricular load leads to the release of BNP.

[0004] Determination of BNP concentration in the blood can provide a lot of useful information for clinical practice. Clinical research on BNP mainly focuses on left ventricular dysfunction (LVD), where left ventricular function refers to contractile function. Whether in normal people or LVD patients, BNP is mainly synthesized and secreted by left ventricular myocardial cells, enters the small veins and returns to the ventricular septal veins and enters the circulation through the coronary sinus. Its secretion is mainly regulated by left ventricular wall tension. The severity of LVD is positively correlated with its secretion. The level of peripheral blood BNP can reflect the ventricular secretion rate and the degree of LVD. More and more literature supports the determination of BNP after myocardial infarction (MI). This can not only identify the presence or absence of left ventricular systolic dysfunction, but also may be superior to cardiac ultrasound diagnosis in judging left ventricular remodeling and the risk of death. In actual clinical work, BNP can also help distinguish asthma caused by heart failure from asthma caused by other reasons. Normal BNP can almost exclude asthma caused by left ventricular dysfunction. Traditionally, long-term monitoring of patients with heart failure is very imperfect. Since BNP has the effects of sodium, diuresis, and vasodilation, it is antagonistic to the activation of the renin-angiotensin-aldosterone system, so it has a large clinical application value. However, BNP has a short biological half-life, and the BNP quality control products of the prior art have the following defects: the first is that the storage period after thawing of the liquid product is short, and the shelf life stability has higher environmental requirements. And the solution storage period after the current freeze-dried reconstitution is shorter. If it is not used up in a short time after reconstitution, it can not be reused, which will cause waste, increase the difficulty of enterprise production and increase the production cost, and bring inconvenience to doctors. The second is that among the existing stabilizers, most of the stability stored in the blood is shorter, and the stabilizer without serum has a larger matrix effect. Therefore, it is urgent to provide a stabilizer that can make BNP stable for a long time. Summary of the invention

[0005] In view of the defects in the prior art, the present invention provides a B-type natriuretic peptide stabilizer and a preparation method and application thereof.

[0006] The present invention provides a B-type natriuretic peptide stabilizer, which comprises the following components:

[0007] 10-90 v / v % human serum matrix, 5-25 g / L trehalose, 10-25 g / L mannitol, 1-5 mM N-acetylcysteine, 1-2.5 g / L fish gelatin, 0.02-0.1 v / v % preservatives and 0.02-0.1 v / v % 5-bromo-5-nitro-1,3-dioxane.

[0008] The human serum stabilizer provided by the present invention is used for the preservation of BNP, which can significantly improve the stability of the chemical properties of BNP, maintain its spatial conformation, and improve its heat resistance. The present invention uses a specific stabilizer to compound with a human serum matrix to avoid the non-specific binding of components in the serum matrix (such as proteins, hormones, immune factors, etc.) with B-type natriuretic peptide, provide protection, and reduce protein denaturation or aggregation.

[0009] The present invention adds a certain amount of fish gelatin to the stabilizer. Fish gelatin has good film-forming property and colloid protection ability in the solution, and can form a protective network around the molecule, thereby preventing the damage caused to BNP by oxidation, light, pH change or other chemical reactions in the outside world, and prolonging the stability of BNP. N-acetylcysteine ​​protects BNP from oxidative damage through antioxidant action, while fish gelatin reduces protein degradation by forming a protective film or micelles. The combination of the two can play a synergistic role by forming a physical barrier and stabilizing the protein solution environment, further improving the stability of B-type natriuretic peptide.

[0010] The invention adds a certain amount of trehalose and mannitol to the stabilizer, and further improves the stability of B-type natriuretic peptide by adjusting the osmotic pressure and hydration of the solution.

[0011] The present invention adds a certain amount of 5-bromo-5-nitro-1,3-dioxane to the stabilizer, which can effectively prolong the storage period of B-type natriuretic peptide, prevent oxidative degradation, and improve stability, especially under high temperature and long-term storage conditions.

[0012] In some embodiments, the preservative is any one of Proclin 300, sodium benzoate, sodium azide, potassium sorbate and 2-methyl-4-isothiazoline-3-one. The preservative can work together with other ingredients to protect the stabilizer formula from microbial contamination, thereby maintaining the safety and stability of BNP.

[0013] In some embodiments, the human serum matrix is ​​negative for four tests including hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody and Treponema pallidum.

[0014] The present invention also provides a method for preparing the B-type natriuretic peptide stabilizer, comprising the following steps:

[0015] S1: weigh mannitol and trehalose by mass and dissolve them in human serum matrix;

[0016] S2: Proclin 300, 5-bromo-5-nitro-1,3-dioxane, N-acetylcysteine ​​and fish gelatin are weighed by mass, added to the solution of S1, and stirred to obtain the B-type natriuretic peptide stabilizer.

[0017] The present invention also provides a B-type natriuretic peptide quality control product, which comprises the B-type natriuretic peptide stabilizer and 80-800 pg / mL B-type natriuretic peptide.

[0018] In some embodiments, the B-type natriuretic peptide quality control product is a lyophilized product.

[0019] The present invention also provides a myocardial detection kit, comprising the B-type natriuretic peptide quality control product.

[0020] The present invention also provides use of the B-type natriuretic peptide stabilizer in preparing a B-type natriuretic peptide quality control product.

[0021] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0022] 1. The stabilizer of the present invention can improve the stability of B-type natriuretic peptide BNP in a serum-containing stabilizer, greatly improve the storage stability, effectively extend the shelf life of independent quality control products in in vitro diagnosis, and improve its commercial value; at the same time, it improves the ability to resist quality change during preparation and storage, and reduces the batch difference caused by the freeze-drying process.

[0023] 2. The present invention significantly improves the disadvantage of too fast degradation of BNP in serum by compounding N-acetylcysteine ​​with fish gelatin. The B-type natriuretic peptide quality control product is reconstituted and stored at 2-8°C with a deviation of less than 10%, and it can be stable for 10 days. Before and after lyophilization, the concentration difference is small, with a deviation of less than 10%. The background test content of the stabilizer is less than the detection limit, and the matrix effect has little effect. After 21 days of acceleration at 37°C, the deviation is less than 20%, and it can be stored at 2-8°C for 3 years. The uniformity after lyophilization is good, and the CV between bottles and the CV within the bottle are both less than 5%. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0026] Example 1

[0027] The components and contents of the BNP stabilizer of the present invention are shown in Table 1:

[0028] Table 1 Composition and content of BNP stabilizer

[0029]

[0030] The preparation steps of BNP stabilizer are as follows:

[0031] 1. Weigh an appropriate amount of human serum matrix, centrifuge at high speed for 5 minutes, take the clarified liquid and perform gradient filtration at 0.45μm and 0.22μm, and store it at 4℃ for later use.

[0032] 2. Weigh NN-acetylcysteine, add Watsons distilled water to dissolve, mix after fully dissolving, filter with 0.22μm filter membrane, and prepare 500mM N-acetylcysteine ​​storage solution. Store at -20℃; keep away from light and avoid repeated freezing and thawing.

[0033] 3. Weigh fish gelatin, add Watsons distilled water and dissolve in a 37°C water bath. Mix well after fully dissolved to prepare 50g / L fish gelatin storage solution for later use.

[0034] 4. Weigh mannitol and trehalose in sequence and dissolve them in the filtered human serum matrix;

[0035] 5. Take Proclin 300 and 5-bromo-5-nitro-1,3-dioxane according to the content of Group A and add them to the solution prepared in step 3. Stir thoroughly to dissolve. After the solution is mixed, measure the pH, filter with a 0.22μm filter membrane, and store at 2~8℃ to prepare Group A stabilizer.

[0036] 6. Take Proclin 300, 5-bromo-5-nitro-1,3-dioxane, and N-acetylcysteine ​​stock solutions according to the content of group B and add them to the solution prepared in step 3. Stir and dissolve them thoroughly. After the solution is mixed, measure the pH, filter it with a 0.22μm filter membrane, and store it at 2~8℃ to prepare group B stabilizer.

[0037] 7. Take Proclin 300, 5-bromo-5-nitro-1,3-dioxane, and fish gelatin storage solution and add them to the solution prepared in step 3 according to the content of group C. Stir thoroughly to dissolve. After the solution is mixed, measure the pH, filter with a 0.22μm filter membrane, and store at 2~8℃ to prepare group C stabilizer.

[0038] 8. Take Proclin 300, 5-bromo-5-nitro-1,3-dioxane, N-acetylcysteine ​​stock solution, and fish gelatin stock solution and add them to the solution prepared in step 3 according to the content of groups D to L, stir thoroughly to dissolve, measure the pH after the solution is mixed, filter with a 0.22μm filter membrane, and store at 2~8℃ to prepare stabilizers of groups D, E, F, G, H, I, J, K, and L.

[0039] Example 2

[0040] In this example, the BNP storage stability of the BNP stabilizer prepared in Example 1 was tested, and the specific steps were as follows:

[0041] The chemiluminescent immunoassay method is used to evaluate the change of BNP activity by the change of luminescence value. The process is as follows: BNP antigen is combined with BNP antibody-coated microparticles, and then acridinium ester-labeled BNP antibody conjugate is added to generate a reaction mixture. Pre-excitation solution and excitation solution are added to the reaction mixture, and the chemiluminescent reaction is measured.

[0042] Expressed as relative luminescence values ​​(RLUs), the BNP antigen concentration value is proportional to the RLUs value.

[0043] Evaluation method: Using the commercialized Human Brain Natriuretic Peptide (BNP) Protein of the brand Tansheng as the raw material, the B-type brain natriuretic peptide assay kit developed by Abbott Laboratories as the detection platform, and the 12 groups of stabilizers prepared in Example 1 were used to prepare low, medium and high levels of BNP quality control products, and the final concentrations were: Level 1 was 80 pg / mL, Level 2 was 320 pg / mL, and Level 3 was 800 pg / mL. The prepared BNP quality control samples were divided into two parts, Group 1 and Group 2. The samples of Group 1 were stored at 2~8°C, and the samples of Group 2 were stored at -20°C after packaging as the control group. They were tested every 2 days, and each sample was tested in parallel 3 times at each time point, and the average was taken. The deviation between the samples of Group 1 and Group 2 was calculated, and the changes in the concentration values ​​of the samples stored at 2~8°C were observed; the results are shown in Table 2 below:

[0044] Table 2 Changes in BNP concentrations of BNP quality control products in groups A to C stored at 2 to 8 °C

[0045]

[0046] Table 3 Changes in BNP concentrations of BNP quality control products in groups D to F stored at 2 to 8 °C

[0047]

[0048] Table 4 Changes in BNP concentration values ​​of BNP quality control products in groups G to I stored at 2 to 8 °C

[0049]

[0050] Table 5 Changes in BNP concentration values ​​of BNP quality control products in groups J-L stored at 2-8℃

[0051]

[0052] From the results in Tables 2-5, it can be seen that after 6 days of placement, the deviation values ​​of groups A-C were all greater than 10%, and after 12 days of placement, the deviation values ​​were around 29%-33%. After 10 days of placement, the deviations of groups E, F, G, H, I, J, K, and L fluctuated around 15%, while group D had excellent stability at 2-8°C after opening the bottle, with a deviation of less than 10% after 10 days of placement, and could be stably placed at 2-8°C for more than 10 days. This indicates that compounding N-acetylcysteine ​​and fish gelatin can significantly improve the stability of BNP quality control products, and the compounding ratio of 5mM N-acetylcysteine ​​and 1g / L fish gelatin is the optimal one.

[0053] Example 3

[0054] Using commercial Human Brain Natriuretic Peptide (BNP) Protein (Tansheng) as raw material, and the B-type brain natriuretic peptide assay kit developed by Abbott Laboratories as the detection platform, the 12 groups of stabilizers prepared in Example 1 were used to prepare BNP quality control products with final concentrations of 80 pg / mL at level 1, 320 pg / mL at level 2, and 800 pg / mL at level 3, and the prepared BNP quality control samples were divided into two parts, group 3 and group 4. Group 3 samples served as the control group and were tested within 1 hour after preparation. Each level of samples was tested in parallel 3 times. Group 4 samples were divided into 100 bottles, each bottle was 2 mL, and the samples of Group 4 were freeze-dried. The freeze-drying procedure was as follows:

[0055] Use a freeze dryer (Jianyi) for freeze drying, primary drying (sublimation drying) and secondary drying (analytical drying). The specific freeze drying parameters are as follows:

[0056] (1) During the cooling process, the temperature drops to 5°C within 5 minutes and is maintained for 60 minutes. There is no specific requirement for the vacuum degree.

[0057] (2) During the cooling process, the temperature drops to 40°C in 5 minutes and is maintained for 120 minutes. There is no specific requirement for the vacuum degree.

[0058] (3) During the heating process, the temperature rises to 5°C in 60 minutes and is maintained for 600 minutes, with a vacuum degree of 6 to 12 Pa;

[0059] (4) The temperature rises to 10°C in 60 minutes and is maintained for 120 minutes, with a vacuum degree of 6 to 12 Pa;

[0060] (5) During the heating process, the temperature rises to 25°C in 60 minutes and is maintained for 360 minutes. The vacuum degree is 11-13 Pa.

[0061] Randomly select 3 bottles of samples with intact morphology after freeze-drying, observe the freeze-drying effect, and reconstitute the freeze-dried BNP quality control product (the solvent is purified water) to the volume before freeze-drying, and then take samples for testing. Each bottle is tested once, the average is taken, and the mean deviation is calculated between the third group of samples and the fourth group of samples to observe the changes in the concentration values ​​of the freeze-dried samples. The results are as follows:

[0062] Freeze-dried form results:

[0063] 1) Freeze-dried morphology standard: The product is qualified if it presents a complete round cake shape after freeze-drying. The qualified rate = number of complete round cake-shaped bottles ÷ total number of freeze-dried bottles * 100%.

[0064] 2) Freeze-dried morphology results: There are 100 bottles of complete round cake-shaped bottles, and the qualified rate is 100%.

[0065] The concentration test results are shown in Table 6 below:

[0066] Table 6 Changes in concentration of BNP freeze-dried quality control after reconstitution

[0067]

[0068] From the results in Table 6, it can be seen that after the freeze-dried reagents prepared in groups A to C were reconstituted, the deviation values ​​of the samples were all greater than 20%, the deviations before and after freeze-drying in groups E, F, G, H, I, J, K, and L were greater than 15% and less than 20%, and the deviation before and after freeze-drying in group D was less than 10%, indicating that the stabilizer prepared in the present application can ensure that the BNP sample is not affected by freeze-drying to the greatest extent.

[0069] Example 4

[0070] The B-type brain natriuretic peptide assay kit developed by Abbott Laboratories was used as the detection platform, and the 12 groups of stabilizers prepared in Example 1 were used as samples to detect the background BNP content experimental group, and the human serum matrix before filtration was used as the control group. The results are shown in Table 7:

[0071] Table 7 BNP background value of BNP stabilizer

[0072]

[0073] From the results in Table 7, it can be seen that the background of the 12 groups of stabilizers prepared in Example 1 are all less than the detection limit, and the matrix effect has little influence.

[0074] Example 5

[0075] Using the commercialized Human Brain Natriuretic Peptide (BNP) Protein of the brand Tansheng as the raw material, the B-type brain natriuretic peptide assay kit developed by Abbott Laboratories as the detection platform, the 12 groups of stabilizers prepared in Example 1 were used to prepare BNP quality control products with a final concentration of 80 pg / mL at level 1, 320 pg / mL at level 2, and 800 pg / mL at level 3, and the prepared BNP quality control products were freeze-dried using a freeze dryer, and primary drying (sublimation drying) and secondary drying (analytical drying) were performed. Specific freeze-drying parameters:

[0076] (1) During the cooling process, the temperature drops to 5°C within 5 minutes and is maintained for 60 minutes. There is no specific requirement for the vacuum degree.

[0077] (2) During the cooling process, the temperature drops to 40°C in 5 minutes and is maintained for 120 minutes. There is no specific requirement for the vacuum degree.

[0078] (3) During the heating process, the temperature rises to 5°C in 60 minutes and is maintained for 600 minutes, with a vacuum degree of 6 to 12 Pa;

[0079] (4) During the heating process, the temperature rises to 10°C in 60 minutes and is maintained for 120 minutes, with a vacuum degree of 6 to 12 Pa;

[0080] (5) During the heating process, the temperature rises to 25°C in 60 minutes and is maintained for 360 minutes. The vacuum degree is 11-13 Pa.

[0081] The freeze-dried BNP quality control products were randomly divided into two parts, group 5 and group 6. The samples in group 5 were used as the control group and stored at 2-8°C. The samples in group 6 were used as the experimental group and stored at 37°C. The samples were tested every 7 days. Each time, each sample was tested in parallel 3 times, and the average was taken. The deviation between the samples in group 5 and group 6 was calculated to observe the changes in the concentration values ​​of the samples stored at 37°C. The results are shown in Table 8 below:

[0082] Table 8 Concentration changes of BNP quality control products

[0083]

[0084] Table 8

[0085]

[0086] Table 8

[0087]

[0088] Table 8

[0089]

[0090] Table 8

[0091]

[0092] Table 8

[0093]

[0094] From the results in Table 8, it can be seen that after accelerated storage at 37°C for 21 days, the deviations of stabilizers in groups A to C were between 26% and 68%, indicating poor stability. After accelerated storage at 37°C for 21 days, the deviations of stabilizers in groups E, F, G, H, I, J, K, and L were between 5% and 25%. After accelerated storage at 37°C for 21 days, the deviation of stabilizers in group D was less than 20%, indicating that stabilizers in group D had better stability at 37°C.

[0095] Example 6

[0096] The B-type brain natriuretic peptide assay kit developed by Abbott Laboratories was used as the detection platform, and the 5th group of BNP quality control products stored at 2-8°C in implementation case 4 were used for detection. Ten bottles of each of the 12 groups of stabilizer samples in group 5 were randomly selected, and each bottle was tested once as the inter-bottle result. Then one bottle was randomly selected from the 10 bottles and repeated for 10 times as the intra-bottle result. The inter-bottle CV and intra-bottle CV were calculated, and the results are shown in Table 9:

[0097] Table 9 Results of inter-bottle CV and intra-bottle CV of BNP stabilizer

[0098]

[0099] From the above results, it can be seen that after freeze-drying, the inter-bottle CV and intra-bottle CV of group D stabilizer were both less than 10%, with better uniformity and less impact from freeze-drying.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A B-type natriuretic peptide quality control product, characterized in that: It comprises a B-type natriuretic peptide stabilizer and 80-800 pg / mL B-type natriuretic peptide, wherein the stabilizer comprises the following components: 10-90 v / v % human serum matrix, 5-25 g / L trehalose, 10-25 g / L mannitol, 5 mM N-acetylcysteine, 1 g / L fish gelatin, 0.02-0.1 v / v % preservatives and 0.02-0.1 v / v % 5-bromo-5-nitro-1,3-dioxane; The B-type natriuretic peptide quality control product is a lyophilized product.

2. The B-type natriuretic peptide quality control product according to claim 1, characterized in that: The preservative is any one of Proclin 300, sodium benzoate, sodium azide, potassium sorbate and 2-methyl-4-isothiazoline-3-one.

3. The B-type natriuretic peptide quality control product according to claim 1, characterized in that: The human serum matrix tested negative for hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody and syphilis spirochete.

4. The B-type natriuretic peptide quality control product according to claim 1, characterized in that: The preparation method of the B-type natriuretic peptide stabilizer comprises the following steps: S1: weigh mannitol and trehalose by mass and dissolve them in human serum matrix; S2: Proclin 300, 5-bromo-5-nitro-1,3-dioxane, N-acetylcysteine ​​and fish gelatin are weighed by mass, added to the solution of S1, and stirred to obtain the B-type natriuretic peptide stabilizer.

5. A myocardial detection kit, characterized in that: Including the B-type natriuretic peptide quality control product described in claim 1.

6. Use of the B-type natriuretic peptide quality control product according to any one of claims 1 to 4 in the preparation of a B-type natriuretic peptide quality control product.

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