Freeze-drying auxiliary materials, freeze-drying preservation solution and use thereof
By using a freeze-drying excipient composed of sugars, amino acids and auxiliaries in a specific ratio, a freeze-drying preservation solution was prepared, which solved the problem of ALP enzyme instability under room temperature and high temperature conditions. This solution enabled long-term preservation of ALP enzyme activity at 50°C and optimized the appearance and hardness of the freeze-dried microspheres, making them suitable for mass production and packaging.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHENZHEN YHLO BIOTECH
- Filing Date
- 2024-11-04
- Publication Date
- 2026-07-30
AI Technical Summary
Existing ALP enzyme reagents are unstable when stored at room temperature and high temperature, which limits their application scenarios, especially in small hospitals or remote areas, and transportation costs are high.
A lyophilization preservation solution was prepared using a specific ratio of sugars, amino acids, and additives. The solution included polyvinylpyrrolidone, polyethylene glycol, mannitol, and bovine serum albumin, along with MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate, and zinc chloride. This solution was used to preserve ALP enzymes.
The freeze-dried preservation solution can be stored for 18 months at room temperature. After storage at high temperature (50℃) for 3 weeks, the enzyme activity retention rate remains above 87%. The freeze-dried microspheres have a smooth appearance and moderate hardness, making them suitable for mass production and packaging.
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Figure CN2024129685_30072026_PF_FP_ABST
Abstract
Description
Lyophilized excipients, lyophilized preservation solutions and their applications Technical Field
[0001] This application relates to the field of biopharmaceutical technology, and in particular to a lyophilized excipient, a lyophilized preservation solution, and their applications. Background Technology
[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. In the in vitro diagnostic field, ALP enzymes are mainly isolated from calf intestinal mucosa and *Escherichia coli*. In clinical testing, such as enzyme-linked immunosorbent assays (ELISA) and enzyme-catalyzed chemiluminescent immunoassays (ACEIs), ALP enzymes and their markers are important components of diagnostic reagents, and their stability is a key factor affecting reagent performance. ALP enzymes have poor thermostability and are extremely sensitive to temperature; high temperatures cause a rapid decrease in ALP enzyme activity.
[0003] In the field of in vitro diagnostics, in-situ lyophilization is commonly used to improve reagent stability. In-situ lyophilization refers to the process of directly lyophilizing liquids within their packaging containers; it is also known as freeze-drying in situ. However, in-situ lyophilization is difficult to implement for large-scale production and redistribution. The fabrication of lyophilized microspheres is a common lyophilized reagent preparation process in recent years. Liquid reagents are dropped into liquid nitrogen using a high-precision dispensing device and rapidly frozen into spheres. Excess liquid is then removed through a suitable lyophilization process. Lyophilized microspheres can retain the activity of enzymes and proteins to the maximum extent, and their porous structure allows for extremely high resolubility, dissolving rapidly upon contact with liquid.
[0004] There are numerous commercially available ALP enzyme liquid stabilizers, most of which claim to be able to be stored for up to a year at 2℃~8℃. However, they cannot actually preserve ALP enzymes at both room temperature and high temperature. For large-scale in vitro diagnostic testing platforms in top-tier hospitals, storing reagents at 2℃~8℃ is acceptable. However, in some smaller hospitals or remote areas with limited economic resources and low daily testing volumes, the low-temperature storage conditions of 2℃~8℃ significantly 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, creating 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 this application provide a lyophilized excipient, a lyophilized preservation solution, and their applications capable of preserving ALP enzymes under high-temperature conditions. The technical solution includes:
[0007] According to a first aspect of this application, a freeze-dried excipient is provided, comprising sugars, amino acids and additives in a mass ratio of (4-20):(2-12):(3-15);
[0008] The adjuvants include one or more of polyvinylpyrrolidone, polyethylene glycol, mannitol, and bovine serum albumin.
[0009] In one embodiment, the mass ratio of the sugars, amino acids and auxiliaries is (4-10):(4-8):(3-12).
[0010] In one embodiment, the sugar includes one or more of trehalose, lactose, sucrose, and inulin.
[0011] In one embodiment, the amino acid includes one or more of alanine, glycine, leucine, lysine, valine, and serine.
[0012] In one embodiment, the polyvinylpyrrolidone includes one or more of polyvinylpyrrolidone K10, polyvinylpyrrolidone K20, polyvinylpyrrolidone K30, polyvinylpyrrolidone K40, polyvinylpyrrolidone K50, polyvinylpyrrolidone K60, polyvinylpyrrolidone K70, polyvinylpyrrolidone K80, and polyvinylpyrrolidone 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 a second aspect of this application, a freeze-dried preservation solution is provided, comprising a freeze-dried storage solution and the aforementioned freeze-dried excipients;
[0015] Based on the total volume of the freeze-dried storage liquid, the concentration of the sugars is 4% (w / v) to 20% (w / v), the concentration of the amino acids is 2% (w / v) to 12% (w / v), and the concentration of the auxiliaries 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 the polyvinylpyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid;
[0018] (2) The concentration of mannitol is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid;
[0019] (3) The concentration of the polyethylene glycol is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid;
[0020] (4) The concentration of bovine serum albumin is 0.5% (w / v) to 10% (w / v) based on the total volume of the freeze-dried storage liquid.
[0021] In one embodiment, the lyophilized stock solution includes MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate, and zinc chloride; the lyophilized stock solution satisfies at least one of the following conditions (1) to (5):
[0022] (1) The concentration of the magnesium chloride hexahydrate is 0.1 mM to 2 mM based on the volume of the MES buffer.
[0023] (2) The concentration of Triton X-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 the calcium chloride dihydrate is 1 mM to 12 mM, based on the volume of the MES buffer solution.
[0026] (5) The concentration of zinc chloride is 0.1 mM to 3 mM based on the volume of MES buffer.
[0027] According to a third aspect of this application, a kit is provided comprising alkaline phosphatase and the above-described lyophilized preservation solution.
[0028] According to the fourth aspect of this application, the use of the lyophilization excipients, the lyophilization preservation solutions, or the kits described above in the preparation of lyophilized microspheres is provided.
[0029] Compared with traditional technologies, this application has the following advantages:
[0030] This application selects specific concentrations of sugars, amino acids, and specific types of excipients as lyophilization excipients. These components work synergistically to prepare a lyophilized preservation solution that, when used to preserve ALP enzyme, can achieve sealed storage at room temperature for more than 18 months, and maintains an enzyme activity retention rate of over 87% after being placed at 50°C for 3 weeks. Furthermore, the lyophilized microspheres prepared using this lyophilized preservation solution are easy to manufacture and package. Attached Figure Description
[0031] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0032] Figure 1 shows the appearance of the freeze-dried microspheres prepared in Examples 2 to 4 and Comparative Example 1 after being placed at room temperature for 18 months. Detailed Implementation
[0033] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this 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 commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.
[0035] In a first aspect, this application provides a freeze-dried excipient comprising sugars, amino acids, and additives in a mass ratio of (4-20):(2-12):(3-15);
[0036] The additives include one or more of polyvinylpyrrolidone, polyethylene glycol, mannitol, and bovine serum albumin.
[0037] Different freeze-drying excipients have a significant impact on the appearance, stability, and hardness of freeze-dried microspheres. Therefore, selecting appropriate freeze-drying excipients is crucial for freeze-dried microspheres.
[0038] Sugars, as important freeze-drying protectants, can protect ALP enzymes from activity loss during freezing and drying. Furthermore, combining sugars with the aforementioned adjuvants can significantly improve the appearance, high-temperature resistance, and hardness of freeze-dried microspheres. Even further, adding specific concentrations of amino acids can significantly improve surface depressions in freeze-dried microspheres, making their surfaces smoother.
[0039] In some embodiments, the mass ratio of sugars, amino acids, and auxiliaries in the above-mentioned freeze-dried excipients is (4-10):(4-8):(3-12). Optionally, the mass ratio of sugars, amino acids, and auxiliaries is (4-8):(6-8):(3-8). Further optionally, the mass ratio of sugars, amino acids, and auxiliaries is 8:8:3.
[0040] In some specific embodiments, the sugars include one or more of trehalose, lactose, sucrose, and inulin.
[0041] In some specific embodiments, the amino acids include one or more of alanine, glycine, leucine, lysine, valine, and serine.
[0042] In some specific embodiments, 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 one optional example, polyvinylpyrrolidone is selected from polyvinylpyrrolidone K40.
[0044] In some specific embodiments, 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 one optional example, polyethylene glycol is selected from polyethylene glycol 20000.
[0046] Understandably, the above-mentioned freeze-drying excipients can be pre-prepared or freshly prepared; the above-mentioned mass ratio is the mass ratio when the three are mixed together.
[0047] In a second aspect, this application provides a lyophilized preservation solution, comprising a lyophilized storage solution and lyophilized excipients of any of the above embodiments; based on the total volume of the lyophilized storage solution, the concentration of sugars 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 excipients 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 auxiliaries is 3% (w / v) to 12% (w / v).
[0049] In some embodiments, the concentration of polyvinylpyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the lyophilized stock. Optionally, the concentration of polyvinylpyrrolidone 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. Optionally, 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. Optionally, 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. Optionally, the concentration of bovine serum albumin is 4% (w / v).
[0053] In some embodiments, the freeze-drying stock solution includes 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 40mM to 60mM; optionally, the concentration of the MES buffer is 45mM to 55mM; further optionally, the concentration of the MES buffer is 50mM.
[0055] In some embodiments, the pH of the MES buffer is 5.8 to 6.2; optionally, the pH of the MES buffer is 5.9 to 6.1; and further optionally, the pH of the MES buffer is 6.0.
[0056] In some specific embodiments, the concentration of magnesium chloride hexahydrate is 0.1 mM to 2 mM based on the volume of the MES buffer; optionally, the concentration of magnesium chloride hexahydrate is 0.4 mM to 0.6 mM; and further optionally, the concentration of magnesium chloride hexahydrate is 0.5 mM.
[0057] In some specific embodiments, the concentration of Triton X-100 is 0.4% (w / v) to 2% (w / v) based on the volume of MES buffer; optionally, the concentration of Triton X-100 is 0.4% (w / v) to 0.6% (w / v); and further optionally, the concentration of Triton X-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; optionally, the concentration of fish skin gelatin is 1.8% (w / v) to 2.2% (w / v); and further optionally, the concentration of fish skin gelatin is 2% (w / v).
[0059] In some specific embodiments, the concentration of calcium chloride dihydrate is 1 mM to 12 mM based on the volume of the MES buffer; optionally, the concentration of calcium chloride dihydrate is 8 mM to 11 mM; further optionally, the concentration of calcium chloride dihydrate is 10 mM.
[0060] In some specific embodiments, the concentration of zinc chloride is 0.1 mM to 3 mM based on the volume of the MES buffer; optionally, the concentration of zinc chloride is 0.5 mM to 0.7 mM; and further optionally, the concentration of zinc chloride is 0.6 mM.
[0061] The freeze-dried preservation solution described in this application is prepared by mixing sugars, amino acids and auxiliaries of specific concentrations and types. The resulting freeze-dried preservation solution can maintain the enzyme activity retention 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, and no shrinkage or melting under high temperature conditions under normal temperature and high temperature conditions.
[0062] In addition, the lyophilized preservation solution of this application has the advantages of simple formulation and low cost.
[0063] A third aspect of this application provides a kit comprising alkaline phosphatase and a lyophilized preservation solution according to any of the above embodiments.
[0064] Understandably, the above-mentioned lyophilized preservation solution can be a pre-prepared lyophilized preservation solution or a freshly prepared lyophilized preservation solution.
[0065] In some embodiments, the kit also includes an anti-human IgE antibody.
[0066] In some specific embodiments, alkaline phosphatase is used to label anti-human IgE antibodies. Optionally, the concentration of alkaline phosphatase-labeled anti-human IgE antibody is 0.7 μg / mL to 0.9 μg / mL, based on the volume of MES buffer; more preferably, the concentration of alkaline phosphatase-labeled anti-human IgE antibody is 0.75 μg / mL to 0.85 μg / mL; even more preferably, the concentration of alkaline phosphatase-labeled anti-human IgE antibody is 0.8 μg / mL.
[0067] Unless otherwise specified, the contents of sugars, amino acids and auxiliaries mentioned above are based on their relative contents when used in combination. However, the form of the freeze-dried excipients provided in this application is not limited to this. The three components are allowed to be placed separately, as long as they can be mixed in the same system when used.
[0068] In a fourth aspect, this application provides the use of the above-mentioned lyophilization excipients, lyophilization preservation solutions, or kits in the preparation of lyophilized microspheres.
[0069] The lyophilized microspheres prepared using the lyophilization preservation solution described in this application have at least the following advantages:
[0070] (1) It alleviates the problem of instability of ALP enzyme and its markers under normal temperature and high temperature conditions: The freeze-dried microspheres of this application can be stored at room temperature for 18 months and maintain ALP enzyme activity of more than 90%.
[0071] (2) The appearance of freeze-dried microspheres has been optimized: the surface of freeze-dried microspheres is smooth without depressions, with moderate hardness, and no shrinkage or melting under high temperature damage conditions of 50℃.
[0072] (3) The opening stability of the freeze-dried microspheres was optimized: the appearance of the freeze-dried microspheres did not change significantly under the conditions of 30℃ and 60% RH.
[0073] (4) It is easy to repackage into single servings, reducing repeated freeze-thaw cycles during use; it can achieve mass production.
[0074] The present application will be further described below with reference to specific embodiments and comparative examples, but should not be construed as limiting the scope of protection of the present application.
[0075] Example 1: Evaluation of the effect of different freeze-dried excipients on the appearance properties of microspheres
[0076] (1) Preparation of freeze-drying preservation solution
[0077] a) Preparation of freeze-dried stock solution
[0078] Based on a pH 6.0 and a concentration of 50 mM MES buffer, 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 and mixed to prepare an ALP enzyme-labeled stock solution.
[0079] The ALP enzyme used in this embodiment was purchased from BBI, catalog number ALPI12G.
[0080] b) Preparation of freeze-dried excipients
[0081] Based on the total volume of the freeze-dried storage liquid prepared in step a), the freeze-dried excipients shown in Table 1 are provided, mixed, and 12 different freeze-dried excipients are prepared.
[0082] Table 1
[0083] c) Add the 12 lyophilized excipients prepared in step b) to the ALP enzyme-labeled storage solution prepared in step a) to prepare the lyophilized preservation solution.
[0084] (2) Preparation of freeze-dried microspheres
[0085] Using the IVEK Digispense 3009 system, the liquid output volume was adjusted to 5 μL. The freeze-dried preservation solution was dropped into a separator containing liquid nitrogen to form frozen microspheres. The frozen microspheres were then transferred to a vacuum freeze dryer pre-frozen to -50°C for freeze-drying. The freeze-drying parameters are shown in Table 2.
[0086] Table 2
[0087] (3) Packaging of freeze-dried microspheres
[0088] After freeze-drying, the freeze-dried microspheres are packaged in a drying chamber with humidity less than 40% and temperature around 25°C.
[0089] (4) Performance evaluation of freeze-dried microspheres
[0090] A portion of the pre-packaged freeze-dried microspheres were placed in a drying oven with humidity less than 40% and temperature of 50℃. After 11 days of accelerated high-temperature degradation, the size change of the freeze-dried microspheres was measured using vernier calipers, and the appearance of the freeze-dried microspheres was observed under a stereomicroscope. The hardness performance was evaluated by drop test. When the freeze-dried microspheres dropped from a height of 1m onto the experimental table, no breakage or powder was recorded as "high hardness", no breakage but powder was recorded as "relatively high hardness", and breakage was recorded as "low hardness".
[0091] Table 3 shows the appearance properties of different freeze-dried excipients after 11 days of accelerated degradation at 50℃. "-" indicates shrinkage, melting, surface depressions, or uneven dimensions, while "+" indicates no shrinkage or melting, a smooth surface without depressions, and uniform dimensions. A "-" or "low" hardness indicates an appearance defect.
[0092] Table 3
[0093] As shown in Groups 1 to 4 of Table 3, adding only sugar as a lyophilization excipient resulted in shrinkage and even melting of the lyophilized microspheres after 11 days of accelerated drying at 50°C. Groups 8 to 11 and Group 1 show that combining trehalose with mannitol, PEG20000, PVPK40, or BSA, respectively, significantly improved the appearance and high-temperature resistance of the lyophilized microspheres to enhance their heat tolerance. Groups 6 to 9 indicate that adding an appropriate concentration of sugar to mannitol or PEG20000 significantly improved the hardness of the lyophilized microspheres. Groups 11 and 12 show that adding an appropriate amount of amino acids improved the surface depressions of the lyophilized microspheres, resulting in a smoother surface.
[0094] The above results indicate that mixing sugars, lyophilization aids, and amino acids at specific concentrations as lyophilization excipients can effectively improve the heat resistance of lyophilized microspheres under high temperature conditions (50℃), maintain a smooth surface appearance, uniform size, and good hardness properties.
[0095] Example 2:
[0096] The steps are basically the same as in Example 1, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 3% (w / v) PVPK40, 8% (w / v) trehalose, and 8% (w / v) alanine.
[0097] The prepared freeze-dried microspheres were placed in a 50℃ drying oven for accelerated degradation for three weeks, and their performance before and after acceleration was evaluated.
[0098] Dissolution rate assessment: Three lyophilized microspheres were placed in each well of an ELISA plate, and 30 μL of ultrapure water was added to each well. Each type of lyophilized microsphere was repeated three times, and the average time for complete dissolution of each type of lyophilized microsphere was recorded.
[0099] Enzyme activity retention rate assessment: Ten lyophilized microspheres were dissolved in 100 μL of ultrapure water, and the alkaline phosphatase activity retention rate (the ratio of activity after accelerated testing at 50℃ to activity after drying at 4℃) was determined using a Mindray fully automated biochemical analyzer and its matching alkaline phosphatase assay kit. The results are shown in Table 4, indicating that the lyophilized microspheres exhibit good enzyme activity retention rate, appearance properties, dissolution rate, and water content under high temperature conditions of 50℃.
[0100] Stability test after opening: After the microspheres were placed at 30℃ 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 in Example 1, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 3% (w / v) PEG20000, 8% (w / v) inulin, and 8% (w / v) glycine.
[0104] The prepared lyophilized microspheres were placed in a 50℃ drying oven for accelerated degradation for three weeks, and their performance before and after acceleration was evaluated. The results are shown in Table 5. After three weeks of accelerated degradation at 50℃, the enzyme activity retention rate of the lyophilized microspheres remained above 90%.
[0105] Stability test after opening: After the microspheres were placed at 30℃ 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 in Example 1, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 12% (w / v) mannitol, 4% (w / v) inulin, and 4% (w / v) alanine.
[0109] The prepared lyophilized microspheres were placed in a 50℃ drying oven for accelerated degradation for three weeks, and their performance before and after acceleration was evaluated. The results are shown in Table 6, indicating that the lyophilized microspheres still maintained good enzyme activity retention, appearance, dissolution rate, and water content after acceleration at 50℃.
[0110] Stability test after opening: After the microspheres were placed at 30℃ 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 in Example 4, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 12% (w / v) mannitol, 4% (w / v) sucrose, and 4% (w / v) alanine; the performance effects are shown in Table 7.
[0114] Table 7
[0115] Comparative Example 1:
[0116] The steps are basically the same as in Example 1, except that the formulation of the freeze-drying excipient is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipient only includes 12% (w / v) PVPK40.
[0117] The performance results are shown in Table 8. With the addition of PVPK40 as a lyophilization excipient, the appearance of the lyophilized microspheres was qualified, but the enzyme activity preservation rate was reduced compared with the other examples. Moreover, the surface of the lyophilized microspheres shrank significantly 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 in Example 2, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 8% (w / v) trehalose, 1% (w / v) alanine and 3% (w / v) PVPK40.
[0121] The performance results are shown in Table 9. Compared with Example 2, the enzyme activity preservation rate of the freeze-dried microspheres in Comparative Example 2 was reduced, and the surface of the microspheres showed depressions.
[0122] Table 9
[0123] Comparative Example 3:
[0124] The steps are basically the same as in Example 2, except that the formulation of the freeze-drying excipients is different: based on the total volume of the freeze-drying storage liquid, the freeze-drying excipients include 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 in Comparative Example 3 was reduced, and the surface of the microspheres began to shrink or melt after two weeks.
[0126] Table 10
[0127] Performance testing
[0128] To verify the stability of the above-mentioned lyophilized excipients for ALP enzyme and its labeling under room temperature conditions, the lyophilized microspheres prepared in Examples 2 to 4 and Comparative Example 1 were placed in sealed glass bottles with desiccant and stored at room temperature (20℃ to 30℃) for 18 months. The enzyme activity retention rate and appearance were then tested. The results are shown in Table 11 and the appearance is shown in Figure 1.
[0129] Table 11
[0130] In summary, the lyophilized microspheres prepared using the specific formulation of the lyophilization preservation solution of this application can maintain stability, good hardness, and satisfactory appearance under high temperature conditions, and still exhibit good enzyme activity retention after being stored at room temperature for 18 months. The lyophilization preservation solution formulation of this application provides a new approach to overcoming the application limitations of alkaline phosphatase at room temperature and in specific environments.
[0131] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.
Claims
1. A freeze-dried excipient, characterized in that, Includes sugars, amino acids, and additives in a mass ratio of (4–20):(2–12):(3–15); The adjuvants include one or more of polyvinylpyrrolidone, polyethylene glycol, mannitol, and bovine serum albumin.
2. The freeze-dried excipient according to claim 1, characterized in that, The mass ratio of the sugars, amino acids and auxiliaries 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 polyvinylpyrrolidone includes one or more of polyvinylpyrrolidone K10, polyvinylpyrrolidone K20, polyvinylpyrrolidone K30, polyvinylpyrrolidone K40, polyvinylpyrrolidone K50, polyvinylpyrrolidone K60, polyvinylpyrrolidone K70, polyvinylpyrrolidone K80 and polyvinylpyrrolidone 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 lyophilized preservation solution, characterized in that, It includes a freeze-dried storage liquid and the freeze-dried excipients according to any one of claims 1 to 6; based on the total volume of the freeze-dried storage liquid, 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 adjuvant is 3% (w / v) to 15% (w / v).
8. The lyophilized preservation solution according to claim 7, characterized in that, The lyophilized preservation solution satisfies at least one of the following conditions (1) to (4): (1) The concentration of the polyvinylpyrrolidone is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid; (2) The concentration of mannitol is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid; (3) The concentration of the polyethylene glycol is 3% (w / v) to 15% (w / v) based on the total volume of the freeze-dried storage liquid; (4) The concentration of bovine serum albumin is 0.5% (w / v) to 10% (w / v) based on the total volume of the freeze-dried storage liquid.
9. The lyophilized preservation solution according to any one of claims 7 to 8, characterized in that, The lyophilized stock solution includes MES buffer, magnesium chloride hexahydrate, Triton X-100, fish skin gelatin, calcium chloride dihydrate, and zinc chloride; the lyophilized stock solution satisfies at least one of the following conditions (1) to (5): (1) The concentration of the magnesium chloride hexahydrate is 0.1 mM to 2 mM based on the volume of the MES buffer. (2) The concentration of Triton X-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 the calcium chloride dihydrate is 1 mM to 12 mM, based on the volume of the MES buffer solution. (5) The concentration of zinc chloride is 0.1 mM to 3 mM based on the volume of MES buffer.
10. A reagent kit, characterized in that, Includes alkaline phosphatase and the lyophilized preservation solution according to any one of claims 7 to 9.
11. The use of the lyophilized excipients 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 the preparation of lyophilized microspheres.