Freeze-drying auxiliary materials, freeze-drying preservation solution and use thereof

Abstract

Provided are freeze-drying auxiliary materials, a freeze-drying preservation solution and the use thereof. The freeze-drying auxiliary materials comprise a carbohydrate, an amino acid and an adjuvant in a mass ratio of (4-20):(2-12):(3-15), wherein the adjuvant comprises one or more of polyvinylpyrrolidone, polyethylene glycol, mannitol and bovine serum albumin. By selecting carbohydrates and amino acids at specific concentrations, along with particular types of adjuvants as the freeze-drying auxiliary materials, where the components cooperate with each other and work together, the resulting freeze-drying preservation solution prepared therefrom, when used for storing an ALP enzyme, can seal and store the ALP enzyme at ambient temperature for 18 months or more, and keep the preservation rate of enzyme activity at 87% or more after the ALP enzyme is placed at the high temperature of 50℃ for 3 weeks.

Description

Freeze-dried excipients, freeze-dried preservation solutions and their applications Technical Field

[0001] The present application relates to the technical field of biological products, and in particular to a freeze-dried excipient, a freeze-dried preservation solution and applications thereof. Background Art

[0002] Alkaline phosphatase (ALP) is widely distributed in animals, plants, and microorganisms. With a molecular weight of approximately 86 kDa, it is a zinc-containing homodimeric protein enzyme. ALP used in in vitro diagnostics is primarily isolated from calf intestinal mucosa and Escherichia coli. In clinical testing, such as enzyme-linked immunosorbent assays (ELISAs) and enzymatic chemiluminescent immunoassays (ELISAs), ALP and its markers are important components of diagnostic reagents, and their stability is a key factor influencing reagent performance. ALP has poor thermal stability and is extremely temperature-sensitive. High temperatures can rapidly reduce ALP activity.

[0003] In the field of in vitro diagnostics, in order to improve the stability of reagents, in situ freeze-drying is usually used to freeze-dry the reagents. In situ freeze-drying refers to the process of placing a liquid in a packaging container and freeze-drying it directly, which is also called in-situ freeze-drying. However, the in situ freeze-drying method is difficult to achieve large-scale production and redistribution. The preparation of freeze-dried microspheres has been a common freeze-dried reagent preparation process in recent years. The liquid detection reagent is dripped into liquid nitrogen through a high-precision dispenser and quickly frozen into balls. The excess liquid is then removed through a reasonable freeze-drying process. Freeze-dried microspheres can maximize the activity of enzymes and proteins, and the microspheres have a loose structure and strong solubility, and can dissolve quickly when encountering liquids.

[0004] There are many types of ALP enzyme liquid stabilizers available, most of which claim to be able to be stored at 2°C to 8°C for up to one year, but they cannot store ALP enzyme at room temperature or high temperature. For large-scale in vitro diagnostic testing platforms in tertiary hospitals, storing reagents at 2°C to 8°C is acceptable. However, in some small hospitals or remote areas with backward economic conditions and small daily test volumes, the low-temperature storage conditions of 2°C to 8°C greatly limit their application scenarios and increase transportation costs. With the development of in vitro diagnostic technologies such as Point of Care Testing (POCT), users have increasingly higher requirements for application scenarios, and there is an urgent need to develop reagents that can be stored and used at room temperature.

[0005] Summary of the Invention

[0006] Based on this, one or more embodiments of the present application provide a freeze-dried excipient, freeze-dried storage solution, and applications thereof capable of preserving ALP enzyme under high temperature conditions. The technical solution includes:

[0007] According to the first aspect of the present application, there is provided a freeze-dried excipient comprising saccharides, amino acids and adjuvants in a mass ratio of (4-20): (2-12): (3-15);

[0008] Wherein, the auxiliary agent includes one or more of polyvinyl pyrrolidone, polyethylene glycol, mannitol and bovine serum albumin.

[0009] In one embodiment, the mass ratio of the carbohydrate, amino acid and auxiliary agent is (4-10): (4-8): (3-12).

[0010] In one embodiment, the sugars include one or more of trehalose, lactose, sucrose and inulin.

[0011] In one embodiment, the amino acids include one or more of alanine, glycine, leucine, lysine, valine and serine.

[0012] In one embodiment, the polyvinyl pyrrolidone includes one or more of polyvinyl pyrrolidone K10, polyvinyl pyrrolidone K20, polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K40, polyvinyl pyrrolidone K50, polyvinyl pyrrolidone K60, polyvinyl pyrrolidone K70, polyvinyl pyrrolidone K80 and polyvinyl pyrrolidone K90.

[0013] In one embodiment, the polyethylene glycol includes one or more of polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000 and polyethylene glycol 20000.

[0014] According to the second aspect of the present application, there is provided a freeze-dried storage solution, comprising a freeze-dried storage solution and the above-mentioned freeze-dried excipient;

[0015] Based on the total volume of the freeze-dried stock solution, the concentration of the sugar is 4% (w / v) to 20% (w / v), the concentration of the amino acid is 2% (w / v) to 12% (w / v), and the concentration of the auxiliary agent is 3% (w / v) to 15% (w / v).

[0016] In one embodiment, the lyophilized preservation solution satisfies at least one of the following conditions (1) to (4):

[0017] (1) The concentration of polyvinylpyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution;

[0018] (2) The concentration of mannitol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution;

[0019] (3) The concentration of the polyethylene glycol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution;

[0020] (4) Based on the total volume of the lyophilized stock solution, the concentration of the bovine serum albumin is 0.5% (w / v) to 10% (w / v).

[0021] In one embodiment, the lyophilized storage solution comprises MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate, and zinc chloride; and the lyophilized storage solution satisfies at least one of the following conditions (1) to (5):

[0022] (1) The concentration of magnesium chloride hexahydrate is 0.1 mM to 2 mM based on the volume of MES buffer;

[0023] (2) The concentration of TritonX-100 is 0.4% (w / v) to 2% (w / v) based on the volume of MES buffer;

[0024] (3) The concentration of the fish skin gelatin is 0.2% (w / v) to 3% (w / v) based on the volume of the MES buffer;

[0025] (4) The concentration of calcium chloride dihydrate is 1 mM to 12 mM based on the volume of MES buffer;

[0026] (5) Based on the volume of the MES buffer, the concentration of zinc chloride is 0.1 mM to 3 mM.

[0027] According to the third aspect of the present application, a kit is provided, comprising alkaline phosphatase and the above-mentioned lyophilized preservation solution.

[0028] According to a fourth aspect of the present application, there is provided a use of the above-mentioned lyophilized excipient, the above-mentioned lyophilized preservation solution or the above-mentioned kit in the preparation of lyophilized microspheres.

[0029] Compared with traditional technologies, this application has the following beneficial effects:

[0030] This application selects specific concentrations of sugars, amino acids, and specific types of adjuvants as lyophilization excipients. These components work together to produce a lyophilized preservation solution that, when used to preserve ALP enzyme, can maintain an enzyme activity retention rate of over 87% after sealed storage at room temperature for over 18 months and at 50°C for three weeks. Furthermore, the lyophilized microspheres produced using this lyophilized preservation solution are easy to produce and package. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0032] FIG1 is a diagram showing the appearance of the freeze-dried microspheres prepared in Examples 2 to 4 of the present application and Comparative Example 1 after being stored at room temperature for 18 months. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotations of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the technical field of this application. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or can be prepared by existing methods.

[0035] In a first aspect of the present application, a freeze-dried excipient is provided, comprising saccharides, amino acids, and auxiliary agents in a mass ratio of (4-20): (2-12): (3-15);

[0036] Wherein, the auxiliary agent includes one or more of polyvinyl pyrrolidone, polyethylene glycol, mannitol and bovine serum albumin.

[0037] Different freeze-drying excipients have a great influence on the appearance, stability, hardness and other properties of freeze-dried microspheres. Therefore, choosing appropriate freeze-drying excipients is crucial for freeze-dried microspheres.

[0038] Sugars act as important lyoprotectants, protecting the ALP enzyme from loss of activity during the freezing and drying process. Furthermore, combining sugars with the aforementioned adjuvants significantly improves the appearance, high-temperature tolerance, and hardness of the freeze-dried microspheres. Furthermore, adding amino acids at specific concentrations can significantly reduce surface concavities, resulting in a smoother surface.

[0039] In some embodiments, the mass ratio of carbohydrates, amino acids, and adjuvants in the above-mentioned lyophilized excipient is (4-10): (4-8): (3-12). Alternatively, the mass ratio of carbohydrates, amino acids, and adjuvants is (4-8): (6-8): (3-8). Further optionally, the mass ratio of carbohydrates, amino acids, and adjuvants is 8:8:3.

[0040] In some embodiments, the sugar comprises one or more of trehalose, lactose, sucrose, and inulin.

[0041] In some embodiments, the amino acid comprises one or more of alanine, glycine, leucine, lysine, valine, and serine.

[0042] In some specific embodiments, the polyvinylpyrrolidone includes one or more of polyvinylpyrrolidone K10 (PVP K10), polyvinylpyrrolidone K20 (PVP K20), polyvinylpyrrolidone K30 (PVP K30), polyvinylpyrrolidone K40 (PVP K40), polyvinylpyrrolidone K50 (PVP K50), polyvinylpyrrolidone K60 (PVP K60), polyvinylpyrrolidone K70 (PVP K70), polyvinylpyrrolidone K80 (PVP K80) and polyvinylpyrrolidone K90 (PVP K90).

[0043] In an optional example, the polyvinyl pyrrolidone is selected from polyvinyl pyrrolidone K40.

[0044] In some specific embodiments, the polyethylene glycol includes one or more of polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, and polyethylene glycol 20000.

[0045] In an optional example, the polyethylene glycol is selected from polyethylene glycol 20000.

[0046] It is understandable that the above-mentioned freeze-dried excipients can be pre-made or ready-mixed; the above-mentioned mass ratio is the mass ratio when the three are mixed.

[0047] The second aspect of the present application provides a freeze-dried preservation solution, comprising a freeze-dried storage solution and a freeze-dried excipient according to any of the above embodiments; based on the total volume of the freeze-dried storage solution, the concentration of carbohydrates is 4% (w / v) to 20% (w / v), the concentration of amino acids is 2% (w / v) to 12% (w / v), and the concentration of the auxiliary agent is 3% (w / v) to 15% (w / v).

[0048] In some embodiments, the concentration of sugars is 4% (w / v) to 10% (w / v), the concentration of amino acids is 4% (w / v) to 8% (w / v), and the concentration of adjuvants is 3% (w / v) to 12% (w / v).

[0049] In some embodiments, the concentration of polyvinyl pyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution. Alternatively, the concentration of polyvinyl pyrrolidone is 3% (w / v).

[0050] In some embodiments, the concentration of mannitol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution. Alternatively, the concentration of mannitol is 12% (w / v).

[0051] In some embodiments, the concentration of polyethylene glycol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution. Alternatively, the concentration of polyethylene glycol is 3% (w / v).

[0052] In some embodiments, the concentration of bovine serum albumin is 0.5% (w / v) to 10% (w / v) based on the total volume of the lyophilized stock solution. Alternatively, the concentration of bovine serum albumin is 4% (w / v).

[0053] In some embodiments, the lyophilized stock solution comprises MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate, and zinc chloride.

[0054] In some specific embodiments, the concentration of the MES buffer is 40 mM to 60 mM; alternatively, the concentration of the MES buffer is 45 mM to 55 mM; further alternatively, the concentration of the MES buffer is 50 mM.

[0055] In some embodiments, the pH value of the MES buffer is 5.8-6.2; alternatively, the pH value of the MES buffer is 5.9-6.1; further alternatively, the pH value of the MES buffer is 6.0.

[0056] In some specific embodiments, based on the volume of MES buffer, the concentration of magnesium chloride hexahydrate is 0.1 mM to 2 mM; alternatively, the concentration of magnesium chloride hexahydrate is 0.4 mM to 0.6 mM; further alternatively, the concentration of magnesium chloride hexahydrate is 0.5 mM.

[0057] In some specific embodiments, the concentration of TritonX-100 is 0.4% (w / v) to 2% (w / v) based on the volume of MES buffer; alternatively, the concentration of TritonX-100 is 0.4% (w / v) to 0.6% (w / v); further alternatively, the concentration of TritonX-100 is 0.5% (w / v).

[0058] In some specific embodiments, the concentration of fish skin gelatin is 0.2% (w / v) to 3% (w / v) based on the volume of MES buffer; alternatively, the concentration of fish skin gelatin is 1.8% (w / v) to 2.2% (w / v); further alternatively, the concentration of fish skin gelatin is 2% (w / v).

[0059] In some specific embodiments, based on the volume of MES buffer, the concentration of calcium chloride dihydrate is 1 mM to 12 mM; alternatively, the concentration of calcium chloride dihydrate is 8 mM to 11 mM; further alternatively, the concentration of calcium chloride dihydrate is 10 mM.

[0060] In some specific embodiments, based on the volume of the MES buffer, the concentration of zinc chloride is 0.1 mM to 3 mM; alternatively, the concentration of zinc chloride is 0.5 mM to 0.7 mM; further alternatively, the concentration of zinc chloride is 0.6 mM.

[0061] The above-mentioned freeze-dried preservation solution of the present application is a mixture of sugars, amino acids and additives of specific concentrations and specific types. The freeze-dried preservation solution prepared can maintain the enzyme activity preservation rate of alkaline phosphatase under normal temperature conditions and even high temperature conditions (such as 50°C); the freeze-dried microspheres prepared further can maintain a smooth surface without depressions, moderate hardness under normal temperature and high temperature conditions, and no shrinkage or melting under high temperature conditions.

[0062] In addition, the freeze-dried preservation solution of the present application has the advantages of simple formula and low cost.

[0063] In a third aspect of the present application, a kit is provided, comprising alkaline phosphatase and the freeze-dried preservation solution of any one of the above embodiments.

[0064] It is understandable that the above-mentioned freeze-dried preservation solution can be a pre-made freeze-dried preservation solution or a freshly prepared freeze-dried preservation solution.

[0065] In some embodiments, the above kit further comprises an anti-human IgE antibody.

[0066] In some embodiments, alkaline phosphatase is used to label anti-human IgE antibodies. Optionally, the concentration of the alkaline phosphatase-labeled anti-human IgE antibody is 0.7 μg / mL to 0.9 μg / mL, based on the volume of MES buffer; further optionally, the concentration of the alkaline phosphatase-labeled anti-human IgE antibody is 0.75 μg / mL to 0.85 μg / mL; further optionally, the concentration of the alkaline phosphatase-labeled anti-human IgE antibody is 0.8 μg / mL.

[0067] In this application, unless otherwise specified, the contents of the above-mentioned sugars, amino acids and adjuvants are based on the relative contents when the three are mixed. However, the form of the freeze-dried excipient provided in this application is not limited to this. The three components can be placed separately as long as they can be mixed in the same system when used.

[0068] The fourth aspect of the present application provides the use of the above-mentioned lyophilized excipient, the above-mentioned lyophilized preservation solution or the kit in the preparation of lyophilized microspheres.

[0069] The freeze-dried microspheres prepared using the freeze-dried preservation solution of the present application have at least the following advantages:

[0070] (1) Alleviating the problem of unstable storage of ALP enzyme and its markers under normal temperature and high temperature conditions: The freeze-dried microspheres of the present application can be stored at room temperature for 18 months and maintain the ALP enzyme activity at more than 90%.

[0071] (2) The appearance of the freeze-dried microspheres was optimized: the surface of the freeze-dried microspheres was smooth without depressions, with moderate hardness, and no shrinkage or melting under high temperature conditions of 50°C.

[0072] (3) The unsealing stability of the freeze-dried microspheres was optimized: under the conditions of 30°C and 60% RH, the appearance of the freeze-dried microspheres did not change significantly.

[0073] (4) It is easy to package into single servings, reducing repeated freezing and thawing during use; it can achieve large-scale production.

[0074] The present application will be further described below with reference to specific embodiments and comparative examples, but they should not be construed as limiting the scope of protection of the present application.

[0075] Example 1: Evaluation of the effects of different lyophilized excipients on the appearance and properties of microspheres

[0076] (1) Preparation of freeze-dried storage solution

[0077] a) Preparation of lyophilized stock solution

[0078] 0.5 mM magnesium chloride hexahydrate, 0.5% (w / v) Triton X-100, 2% (w / v) fish skin gelatin, 10 mM calcium chloride dihydrate, 0.6 mM zinc chloride, and 0.8 μg / mL ALP enzyme-labeled anti-human IgE marker were provided in a 50 mM MES buffer with a pH of 6.0 and mixed to prepare an ALP enzyme marker stock solution.

[0079] The ALP enzyme used in this example was purchased from BBI with the product number ALPI12G.

[0080] b) Preparation of lyophilized excipients

[0081] Based on the total volume of the lyophilized stock solution prepared in step a), the lyophilized excipient raw materials shown in Table 1 were provided and mixed to prepare 12 lyophilized excipients with different formulations.

[0082] Table 1

[0083] c) adding the 12 freeze-dried excipients prepared in step b) into the ALP enzyme marker stock solution prepared in step a) to prepare a freeze-dried storage solution.

[0084] (2) Preparation of freeze-dried microspheres

[0085] Using the IVEK Digispense 3009 system, the output volume was adjusted to 5 μL, and the lyophilized storage solution was dropped into a separator containing liquid nitrogen to form frozen microspheres. The frozen microspheres were then transferred to a vacuum freeze dryer that had been pre-frozen to -50°C for freeze drying. The freeze drying parameters are shown in Table 2.

[0086] Table 2

[0087] (3) Freeze-dried microspheres packaging

[0088] After freeze drying, the freeze-dried microspheres are packaged in a drying room with a humidity of less than 40% and a temperature of about 25°C.

[0089] (4) Performance evaluation of freeze-dried microspheres

[0090] A portion of the packaged freeze-dried microspheres were placed in a drying oven with a humidity of less than 40% and a temperature of 50°C. After 11 days of high-temperature accelerated destruction, the size changes of the freeze-dried microspheres were measured using a vernier caliper, and the appearance of the freeze-dried microspheres was observed under a stereo microscope. The hardness performance was evaluated through a drop test. When the freeze-dried microspheres were dropped from a height of 1 m onto the experimental table, no breakage or powdering was recorded as "high hardness", no breakage but powdering was recorded as "relatively high hardness", and breakage was recorded as "low".

[0091] Table 3 shows the appearance of various lyophilized excipients after accelerated destruction at 50°C for 11 days. "-" indicates shrinkage, melting, surface depressions, or dimensional inhomogeneity, while "+" indicates no shrinkage or melting, a smooth surface without depressions, and uniform size. A "-" or "low" hardness rating indicates an unacceptable appearance.

[0092] Table 3

[0093] As can be seen from Groups 1 to 4 in Table 3, when only sugar was added as a freeze-drying excipient, the freeze-dried microspheres shrank or even melted after 11 days of accelerated storage at 50°C. As can be seen from Groups 8 to 11 and Group 1, in order to improve the high temperature tolerance of sugar, trehalose was combined with mannitol, PEG20000, PVPK40 or BSA, respectively, which can significantly improve the appearance and high temperature tolerance of the freeze-dried microspheres. As can be seen from Groups 6 to 9, adding an appropriate concentration of sugar to mannitol or PEG20000 can significantly improve the hardness of the freeze-dried microspheres. As can be seen from Groups 11 and 12, adding an appropriate amount of amino acids can improve the concave surface of the freeze-dried microspheres, making the surface of the freeze-dried microspheres smoother.

[0094] The above results show that mixing specific concentrations of sugars, freeze-drying aids and amino acids as freeze-drying excipients can effectively improve the heat resistance of freeze-dried microspheres under high temperature conditions (50°C), maintain a smooth surface, uniform size and good hardness.

[0095] Example 2:

[0096] The steps are basically the same as those in Example 1, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 3% (w / v) PVPK40, 8% (w / v) trehalose, and 8% (w / v) alanine.

[0097] The prepared freeze-dried microspheres were placed in a drying oven at 50°C for three weeks for accelerated destruction, and the performance before and after acceleration was evaluated.

[0098] Dissolution rate assessment: Place three freeze-dried microspheres in the wells of an ELISA plate and add 30 μL of ultrapure water to each well. Repeat this procedure three times for each type of freeze-dried microsphere and record the average time it takes for each freeze-dried microsphere to completely dissolve.

[0099] Enzyme activity preservation evaluation: Ten lyophilized microspheres were dissolved in 100 μL of ultrapure water. The alkaline phosphatase activity preservation (ratio of activity after 50°C accelerated testing to activity after 4°C dry storage) was measured using a Mindray fully automated biochemical analyzer and the accompanying alkaline phosphatase assay kit. The results, shown in Table 4, demonstrate that the lyophilized microspheres exhibited excellent enzyme activity preservation, appearance, dissolution rate, and moisture content at 50°C.

[0100] Unsealed stability test: After the microspheres were placed at 30°C and 60% RH for 3 hours, there was no obvious shrinkage on the surface of the microspheres.

[0101] Table 4

[0102] Example 3:

[0103] The steps are basically the same as those in Example 1, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 3% (w / v) PEG20000, 8% (w / v) inulin, and 8% (w / v) glycine.

[0104] The prepared lyophilized microspheres were placed in a 50°C drying oven for three weeks for accelerated destruction, and their performance before and after acceleration was evaluated. The results are shown in Table 5. After three weeks of accelerated destruction at 50°C, the enzyme activity preservation rate of the lyophilized microspheres remained above 90%.

[0105] Unsealed stability test: After the microspheres were placed at 30°C and 60% RH for 3 hours, there was no obvious shrinkage on the surface of the microspheres.

[0106] Table 5

[0107] Example 4:

[0108] The steps are basically the same as those in Example 1, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 12% (w / v) mannitol, 4% (w / v) inulin, and 4% (w / v) alanine.

[0109] The prepared freeze-dried microspheres were placed in a 50°C drying oven for three weeks for accelerated destruction, and their performance before and after acceleration was evaluated. The results, shown in Table 6, demonstrate that the freeze-dried microspheres maintained good enzyme activity preservation, appearance, dissolution rate, and moisture content after accelerated exposure to 50°C.

[0110] Unsealed stability test: After the microspheres were placed at 30°C and 60% RH for 3 hours, there was no obvious shrinkage on the surface of the microspheres.

[0111] Table 6

[0112] Example 5:

[0113] The results are basically the same as those in Example 4, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 12% (w / v) mannitol, 4% (w / v) sucrose, and 4% (w / v) alanine; the performance results are shown in Table 7.

[0114] Table 7

[0115] Comparative Example 1:

[0116] The steps are basically the same as those in Example 1, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient only includes 12% (w / v) PVPK40.

[0117] The performance results are shown in Table 8. When only PVPK40 was added as a lyophilization excipient, the appearance of the lyophilized microspheres was qualified, but the enzyme activity preservation rate was reduced compared with the examples; and the surface of the lyophilized microspheres showed obvious shrinkage after being placed at 30°C and 60% RH for 3 hours.

[0118] Table 8

[0119] Comparative Example 2:

[0120] The steps are basically the same as those in Example 2, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 8% (w / v) trehalose, 1% (w / v) alanine, and 3% (w / v) PVPK40.

[0121] The performance effects are shown in Table 9. Compared with Example 2, the enzyme activity preservation rate of the freeze-dried microspheres of Comparative Example 2 is reduced, and depressions appear on the surface of the microspheres.

[0122] Table 9

[0123] Comparative Example 3:

[0124] The steps are basically the same as those in Example 2, except that the formula of the lyophilized excipient is different: based on the total volume of the lyophilized stock solution, the lyophilized excipient includes 8% (w / v) trehalose, 8% (w / v) alanine and 1% (w / v) PVPK40.

[0125] The performance results are shown in Table 10. Compared with Example 2, the enzyme activity preservation rate of the freeze-dried microspheres of Comparative Example 3 is reduced, and the surface of the microspheres begins to shrink or melt after two weeks.

[0126] Table 10

[0127] Performance Testing

[0128] To verify the storage stability of the above-mentioned lyophilized excipients for ALP enzyme and its marker at room temperature, the lyophilized microspheres prepared in Examples 2 to 4 and Comparative Example 1 were placed in sealed glass bottles containing a desiccant and stored at room temperature (20°C to 30°C) for 18 months. The enzyme activity retention rate and appearance were tested. The results are shown in Table 11, and the appearance is shown in Figure 1.

[0129] Table 11

[0130] In summary, the freeze-dried microspheres prepared using the freeze-dried preservation solution with the specific formulation of this application can maintain stability, good hardness, and acceptable appearance under high temperature conditions, and still have good enzyme activity retention after 18 months at room temperature. The freeze-dried preservation solution formulation of this application provides new ideas for breaking through the application limitations of alkaline phosphatase at room temperature and in scenarios.

[0131] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present application shall be determined by the appended claims.

Claims

1. A freeze-dried excipient, characterized in that: The invention comprises saccharides, amino acids and auxiliary agents in a mass ratio of (4-20): (2-12): (3-15); Wherein, the auxiliary agent includes one or more of polyvinyl pyrrolidone, polyethylene glycol, mannitol and bovine serum albumin.

2. The freeze-dried excipient according to claim 1, characterized in that The mass ratio of the sugar, amino acid and auxiliary agent is (4-10): (4-8): (3-12).

3. The freeze-dried excipient according to any one of claims 1 to 2, characterized in that The sugars include one or more of trehalose, lactose, sucrose and inulin.

4. The freeze-dried excipient according to any one of claims 1 to 2, characterized in that The amino acids include one or more of alanine, glycine, leucine, lysine, valine and serine.

5. The freeze-dried excipient according to any one of claims 1 to 2, characterized in that The polyvinyl pyrrolidone includes one or more of polyvinyl pyrrolidone K10, polyvinyl pyrrolidone K20, polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K40, polyvinyl pyrrolidone K50, polyvinyl pyrrolidone K60, polyvinyl pyrrolidone K70, polyvinyl pyrrolidone K80 and polyvinyl pyrrolidone K90.

6. The freeze-dried excipient according to any one of claims 1 to 2, characterized in that The polyethylene glycol includes one or more of polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000 and polyethylene glycol 20000.

7. A freeze-dried preservation solution, characterized in that The invention comprises a freeze-dried stock solution and the freeze-dried excipient according to any one of claims 1 to 6; based on the total volume of the freeze-dried stock solution, the concentration of the carbohydrate is 4% (w / v) to 20% (w / v), the concentration of the amino acid is 2% (w / v) to 12% (w / v), and the concentration of the auxiliary agent is 3% (w / v) to 15% (w / v).

8. The freeze-dried preservation solution according to claim 7, characterized in that The freeze-dried preservation solution satisfies at least one of the following conditions (1) to (4): (1) The concentration of polyvinylpyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution; (2) The concentration of mannitol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution; (3) The concentration of the polyethylene glycol is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock solution; (4) Based on the total volume of the lyophilized stock solution, the concentration of the bovine serum albumin is 0.5% (w / v) to 10% (w / v).

9. The freeze-dried storage solution according to any one of claims 7 to 8, characterized in that The freeze-dried storage solution comprises MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate and zinc chloride; the freeze-dried storage solution satisfies at least one of the following conditions (1) to (5): (1) The concentration of magnesium chloride hexahydrate is 0.1 mM to 2 mM based on the volume of MES buffer; (2) The concentration of TritonX-100 is 0.4% (w / v) to 2% (w / v) based on the volume of MES buffer; (3) The concentration of the fish skin gelatin is 0.2% (w / v) to 3% (w / v) based on the volume of the MES buffer; (4) The concentration of calcium chloride dihydrate is 1 mM to 12 mM based on the volume of MES buffer; (5) Based on the volume of the MES buffer, the concentration of zinc chloride is 0.1 mM to 3 mM.

10. A kit, characterized in that The invention comprises alkaline phosphatase and the freeze-dried storage solution according to any one of claims 7 to 9.

11. Use of the lyophilized excipient according to any one of claims 1 to 6, the lyophilized preservation solution according to any one of claims 7 to 9, or the kit according to claim 10 in preparing lyophilized microspheres.

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