Recombinant tachypleus amebocyte lyophilized preparation for bacterial endotoxin detection and preparation method and application thereof

Abstract

The invention discloses a recombinant tachypleus amebocyte lyophilized preparation for bacterial endotoxin detection as well as a preparation method and application of the recombinant tachypleus amebocyte lyophilized preparation. The recombinant tachypleus amebocyte lyophilized preparation is obtained by lyophilizing a lyophilized solution, wherein the lyophilized solution comprises the following components: mannitol, cane sugar, polyvinylpyrrolidone, recombinant human albumin, ascorbic acid, sodium chloride, inositol, L-arginine hydrochloride, ethylene glycol diethyl ether diamine tetraacetic acid, Tween 80, Proclin 300, Tris-HCl with the pH value of 7.0-8.0, a recombinant tachypleus amebocyte lyophilized preparation, a chromogenic substrate and a reaction buffer solution. The invention further discloses a preparation method and application of the recombinant tachypleus amebocyte lyophilized preparation. The recombinant tachypleus amebocyte lyophilized preparation is prepared by matching a multi-element synergistic protection system with a seven-stage gradient temperature control freeze-drying process, and can be used for high-sensitivity and high-stability detection of bacterial endotoxin in drugs, medical instruments, biological products and clinical samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, it relates to a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection, its preparation method, and its application. Background Technology

[0002] Bacterial endotoxin detection is a crucial step in the quality control of pharmaceuticals and medical devices. Traditional horseshoe crab reagents rely on amoeboid cell lysates extracted from the blood of endangered horseshoe crabs, which presents challenges such as animal protection controversies, significant batch-to-batch variations, potential viral contamination risks, and limitations imposed by natural resources. These issues make it difficult to meet the urgent need in modern biomedicine for highly consistent, animal-free reagents.

[0003] To protect horseshoe crab resources and improve detection reliability, recombinant horseshoe crab reagent technology based on genetic engineering has emerged and has been approved by the United States Pharmacopeia (USP). <86> The recombinant horseshoe crab (LCL) reagent is included in the Japanese Pharmacopoeia (JP18) and the European Pharmacopoeia (EP 2.6.32), among others. Currently, there are two main types: single-factor recombinant factor C (rFC) and three-factor recombinant horseshoe crab reagent (rCR) containing recombinant factor C, factor B, and coagulase. The latter mimics the natural cascade reaction pathway of traditional horseshoe crab reagents, offering advantages such as high detection accuracy, good sensitivity, and strong anti-interference capabilities, thus having a broader application prospect. However, the development of rCR formulations faces significant stability challenges. Unlike rFC, which exhibits certain stability under specific protective liquid conditions, the recombinant coagulase in rCR has a complex structure and is thermodynamically unstable. Furthermore, commonly used chromogenic substrates (such as peptide chains containing p-nitroaniline groups) are prone to hydrolysis or oxidative degradation in aqueous solutions. This results in extremely short shelf life and rapid decline in biological activity for liquid rCR formulations, severely limiting their commercial application and ease of transportation. Therefore, developing lyophilized rCR formulations has become a key pathway to improve their storage stability and feasibility.

[0004] Existing freeze-drying processes often employ a simple "pre-freezing followed by single-temperature plateau sublimation" model (e.g., pre-freezing at -40℃, followed by isothermal sublimation at -20℃). This crude approach struggles to address the complex thermodynamic requirements of multi-component protein systems during freeze-drying. Such processes are prone to uneven ice crystal growth, localized overheating, or concentrated drying stress, leading to protein denaturation and aggregation, freeze-dried cake structural collapse, or difficulty in reconstitution. Furthermore, conventional cryoprotectant systems typically use only a single excipient (e.g., sucrose or mannitol alone). While these can form a glassy matrix, they are still susceptible to excipient crystallization, moisture reabsorption, or protein aggregation during long-term storage or under high temperature and humidity conditions, making it difficult to simultaneously maintain protein activity, reconstitution rate, and long-term storage stability. In addition, some existing formulations contain excessively high concentrations of surfactants or excipients, which may interfere with detection signals or affect clinical compatibility.

[0005] Therefore, there is an urgent need for a synergistic protective lyophilization solution formulation targeting the characteristics of the three-factor recombinant horseshoe crab reagent, along with a refined gradient temperature-controlled lyophilization process. This would ensure rapid and complete reconstitution while significantly improving the physicochemical stability of the lyophilized product, achieving a shelf life of no less than 12 months at 2-8°C, and meeting the stringent requirements of high reproducibility, high sensitivity, and long-term reliability for endotoxin detection in pharmaceuticals and medical devices. Summary of the Invention

[0006] The first objective of this invention is to provide a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection, which contains a specific ratio of multi-component stable system that can effectively protect the activity of recombinant proteins, inhibit the degradation of chromogenic substrates, and ensure that the lyophilized cake has a loose structure and rapid reconstitution.

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned recombinant horseshoe crab reagent lyophilized formulation, which utilizes a staged gradient temperature-controlled lyophilization process to minimize protein denaturation and structural collapse by precisely controlling the freezing rate, sublimation temperature and desorption drying gradient.

[0008] The third objective of this invention is to provide the application of the above-mentioned recombinant horseshoe crab reagent lyophilized formulation in the preparation of reagents for bacterial endotoxin detection, wherein the reagent is physicochemically stable and has excellent detection performance, possessing high sensitivity, high reproducibility and long-term storage stability.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection. The recombinant horseshoe crab reagent lyophilized formulation is obtained by lyophilizing a lyophilizing solution, which is prepared from the following components at the following final concentrations: Mannitol 5-10%, sucrose 2-5%, polyvinylpyrrolidone 1-3%, recombinant human serum albumin (rHSA) 5-10 mg / mL, ascorbic acid 0.1-0.5%, sodium chloride 0.8-1.0%, inositol 2-5%, L-arginine hydrochloride 1-2%, ethylene glycol diethyl ether diaminetetraacetic acid 0.1-0.5 mM, Tween 80 0.05-0.1%, Proclin 300 0.1-0.3%, Tris-HCl 50-100 mM (pH 7.0-8.0), recombinant horseshoe crab reagent 0.1-5.0 μg / mL, chromogenic substrate 0.1-1.0 mM, and reaction buffer; % is the weight-volume percentage (w / v), and the unit of the weight-volume percentage is g / mL.

[0010] In a specific embodiment of the present invention, the final concentrations of each component of the reaction buffer in the lyophilized solution are: magnesium acetate 1-10 mM, polysorbate-20 0.01-0.05%, and hydroxypropyl-β-cyclodextrin 0.1-1 mM; % is the weight-volume percentage (w / v), and the unit of the weight-volume percentage is g / mL. It can be prepared by methods already disclosed in the prior art or purchased commercially.

[0011] In a specific embodiment of the present invention, the recombinant horseshoe crab reagent is a recombinant three-factor protein, including recombinant factor C, recombinant factor B, and recombinant coagulase; wherein, the recombinant factor C, recombinant factor B, and recombinant coagulase can be prepared using methods disclosed in the prior art or purchased commercially.

[0012] In a specific embodiment of the present invention, the chromogenic substrate is a horseshoe crab tripeptide-pNA chromogenic substrate.

[0013] In a specific embodiment of the present invention, the final concentration of sodium chloride in the lyophilization solution is 0.9%.

[0014] In a specific embodiment of the present invention, the polyvinylpyrrolidone is PVP K30.

[0015] In a specific embodiment of the present invention, the preparation method of the lyophilization solution is as follows: Prepare a recombinant human serum albumin stock solution, filter and sterilize for later use; Weigh mannitol, sucrose, polyvinylpyrrolidone, ascorbic acid, sodium chloride, inositol and L-arginine hydrochloride, dissolve them in sterile water for injection, and add Tris-HCl, EGTA, Tween 80, Proclin 300 and the recombinant human serum albumin stock solution at pH 7.0-8.0. Add recombinant horseshoe crab reagent, chromogenic substrate, and reaction buffer; The mixture was brought to a final volume, mixed thoroughly, and then filtered to remove bacteria.

[0016] The present invention further provides a method for preparing the above-mentioned recombinant horseshoe crab reagent lyophilized formulation, comprising the following steps: The above-mentioned lyophilized liquid was dispensed into pyrogen-free containers and placed in a freeze dryer. The freeze-drying process was carried out under vacuum conditions of 0.08 ~ 0.1 mbar according to the following procedure: Cool to -50~-20℃, keep warm for 2-4 hours, and quickly pre-freeze to form fine ice crystals; Continue freezing at -50℃ for 3-5 hours to ensure complete curing; Heat to -45~-42℃ and hold for 10-12 h to achieve primary sublimation and slow release of free water. Heat to -40~-34℃ and hold for 8-10 hours. During the main sublimation stage, free water is efficiently removed. Heat to -35~-26℃ and hold for 6-8 hours to allow residual ice crystals to sublimate and prevent collapse; Heat to +25℃ and keep warm for 4-6 hours to desorb and dry in the initial stage, removing non-freezing water. Heat to +30℃, keep warm for 2-4 hours, and finally dry to remove as much moisture as possible; After freeze-drying, stopper and cap the product to obtain the recombinant horseshoe crab reagent freeze-dried formulation.

[0017] In a specific embodiment of the present invention, the pyrogen-free container is a pyrogen-free vial.

[0018] The present invention also provides the application of the above-mentioned recombinant horseshoe crab reagent lyophilized formulation in the detection of bacterial endotoxins and / or the preparation of reagents for the detection of bacterial endotoxins.

[0019] The present invention further provides a kit for detecting bacterial endotoxins, the kit comprising the above-mentioned recombinant horseshoe crab reagent lyophilized formulation.

[0020] The beneficial effects of this invention are as follows: The recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection of this invention is prepared using a multi-component synergistic protection system combined with a seven-stage gradient temperature-controlled lyophilization process. It exhibits good physical stability and high retention of biological activity, making it suitable for high-sensitivity and high-stability detection of bacterial endotoxins in pharmaceuticals, medical devices, biological products, and clinical samples. It has significant potential for widespread application, as detailed below: 1. Formula Synergistic Protection System Synergistic effect of glassy matrix: Mannitol provides a rigid framework to prevent collapse, while sucrose forms a high-viscosity glassy state in the amorphous region. The two work together to inhibit the thermal motion of protein molecules during freeze-drying and storage, thus maintaining the tertiary structure.

[0021] Dual protection against interface and oxidation: rHSA preferentially adsorbs at the gas-liquid interface to prevent protein denaturation caused by surface tension; PVP K30 not only increases solution viscosity to inhibit ice crystal growth, but also synergistically enhances the mechanical strength of the freeze-dried cake with rHSA; ascorbic acid, as a strong reducing agent, specifically scavenge dissolved oxygen and free radicals, blocking the oxidative degradation pathway of the chromogenic substrate.

[0022] Stable microenvironment: EGTA chelates metal ions to prevent activation of proteolytic enzymes; Tween 80 reduces interfacial tension and accelerates resolution; Proclin 300 provides broad-spectrum antibacterial protection; Sodium chloride maintains an isotonic environment and reduces resolution shock; Inositol makes the freeze-dried cake loose and porous, which can increase the rate of water penetration and accelerate resolution; L-arginine hydrochloride can prevent protein from instantaneously agglomerating and precipitating during resolution.

[0023] 2. Gradient freeze-drying to prevent collapse This invention employs an innovative seven-stage temperature control curve for freeze-drying: a two-stage pre-freezing process at -50℃ ensures complete crystallization; slow sublimation at -45~-42℃ to 35~-26℃ precisely matches the eutectic point; and stepwise desorption at 25℃ / 30℃ completely removes bound water without causing thermal degradation. This effectively and significantly avoids the protein denaturation and structural collapse caused by traditional single-platform sublimation, resulting in more uniform and reliable freeze-dried formulations.

[0024] 3. Excellent performance The lyophilized formulation of this invention is a white, loose, blocky substance with a reconstitution time of ≤20 seconds. Experimental data show that after storage at 2-8℃ for 12 months, the activity retention rate is ≥85%, the endotoxin detection limit reaches 0.005 EU / mL, and the linear correlation coefficient R²>0.998, which is far superior to existing technologies.

[0025] 4. No animal-derived risk The core components of the freeze-dried formulation of this invention are all chemically synthesized or recombinantly expressed, without involving any animal-derived components. This fully complies with the regulatory requirements for animal-free reagents, avoids ethical and resource limitations, and has higher promotional value and safety. Detailed Implementation

[0026] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0027] All raw materials used in this invention are cell culture grade or pharmaceutical grade and are stored according to relevant requirements. Unless otherwise specified, the experimental methods in the following examples are conventional methods.

[0028] Example 1: Preparation of a lyophilized recombinant horseshoe crab reagent for bacterial endotoxin detection I. Preparation of lyophilization solution Prepare a 200 mg / mL recombinant human serum albumin (rHSA) stock solution, and sterilize it by 0.1 μm filtration for later use; Weigh out 10.0 g of mannitol, 5.0 g of sucrose, 1.0 g of polyvinylpyrrolidone (PVP K30), 0.1 g of ascorbic acid, 0.9 g of sodium chloride, 2.0 g of inositol, and 2.0 g of L-arginine hydrochloride. Dissolve them in sterile water for injection, and add Tris-HCl (pH 7.0), EGTA, Tween 80, Proclin 300, and the above-mentioned rHSA stock solution to the final concentrations of 50 mM, 0.5 mM, 0.05% (w / v), 0.3% (w / v), and 10 mg / mL, respectively. Add the recombinant three-factor protein mixture (containing recombinant factor C, recombinant factor B, and recombinant coagulase, prepared according to patent CN105866080B, with a mass ratio of 1:1:1, to a final concentration of 1.0 μg / mL), horseshoe crab tripeptide-pNA chromogenic substrate (to a final concentration of 0.5 mM), and reaction buffer (prepared according to patent CN121612672A, with components and final concentrations of 5 mM magnesium acetate, 0.01% (w / v) polysorbate-20, and 0.5 mM hydroxypropyl-β-cyclodextrin). Bring the volume to 100 mL, mix well, and then filter through a 0.1 μm filter for sterilization.

[0029] The filtrate was dispensed into pyrogen-free vials, 0.5 mL per vial, to obtain a lyophilized solution. The final concentrations of each component in the lyophilized solution were as follows: mannitol 10% (w / v), sucrose 5% (w / v), PVP K30 1% (w / v), ascorbic acid 0.1% (w / v), sodium chloride 0.9% (w / v), inositol 2% (w / v), L-arginine hydrochloride 2% (w / v), Tris-HCl 50 mM (pH 7.0), EGTA 0.5 mM, Tween 80 0.05% (w / v), Proclin 300 0.3% (w / v), recombinant human serum albumin (rHSA) 10 mg / mL, recombinant factor three protein 1.0 μg / mL, horseshoe crab tripeptide-pNA chromogenic substrate 0.5 mM, magnesium acetate 5 mM, and polysorbate-20. 0.01% (w / v), 0.5 mM hydroxypropyl-β-cyclodextrin; the unit of w / v is g / mL; II. Lyophilization to obtain recombinant horseshoe crab reagent lyophilized formulation Place the freeze-drying solution on the freeze dryer shelf and run the following program for freeze-drying: Cool to -50℃ and keep warm for 2 hours; Maintain at -50℃ for 3 hours; Heat to -42℃ and hold for 10 hours; Heat to -34℃ and hold for 8 hours; Heat to -26℃ and hold for 6 hours; Heat to +25℃ and hold for 4 hours; Heat to +30℃ and hold for 2 hours; The vacuum level was controlled at 0.08–0.1 mbar throughout the process; After lyophilization, stopper and cap to obtain the recombinant horseshoe crab reagent lyophilized preparation for endotoxin detection.

[0030] Example 2: Preparation of a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection The amounts of each component were adjusted (mannitol 5.0 g, sucrose 2.0 g, PVP K30 2.0 g, ascorbic acid 0.5 g, sodium chloride 0.9 g, inositol 5.0 g, L-arginine hydrochloride 2.0 g), otherwise the same as in Example 1, so that the final concentrations of each component in the lyophilized solution were: mannitol 5% (w / v), sucrose 2% (w / v), PVP K30 2% (w / v), ascorbic acid 0.5% (w / v), sodium chloride 0.9% (w / v), inositol 5.0% (w / v), L-arginine hydrochloride 2.0% (w / v), Tris-HCl (pH 7.5) 100 mM, EGTA 0.1 mM, Tween 80 0.1% (w / v), Proclin 300 0.1% (w / v), rHSA 5 mg / mL, and recombinant factor three protein 1.0 g. μg / mL, horseshoe crab tripeptide-pNA chromogenic substrate 0.5 mM, magnesium acetate 5 mM, polysorbate-20 0.01% (w / v), hydroxypropyl-β-cyclodextrin 0.5 mM; the unit of w / v is g / mL; II. Lyophilization to obtain recombinant horseshoe crab reagent lyophilized formulation Place the freeze-drying solution on the freeze dryer shelf and run the following program for freeze-drying: Cool to -20℃ and keep warm for 4 hours; Cool to -50℃ and keep warm for 5 hours; Heat to -44℃ and hold for 12 hours; Heat to -38℃ and hold for 10 hours; Heat to -32℃ and hold for 8 hours; Heat to +25℃ and hold for 6 hours; Heat to +30℃ and hold for 4 hours; The vacuum level was controlled at 0.08–0.1 mbar throughout the process; After lyophilization, stopper and cap to obtain the recombinant horseshoe crab reagent lyophilized preparation for endotoxin detection.

[0031] Example 3: Preparation of a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection The amounts of each component were adjusted (mannitol 5.0 g, sucrose 5.0 g, PVP K30 3.0 g, ascorbic acid 0.5 g, sodium chloride 0.9 g, inositol 2.0 g, L-arginine hydrochloride 1.0 g), otherwise the same as in Example 1, so that the final concentrations of each component in the lyophilized solution were: mannitol 5% (w / v), sucrose 5% (w / v), PVP K30 3% (w / v), ascorbic acid 0.5% (w / v), sodium chloride 0.9% (w / v), inositol 2% (w / v), L-arginine (hydrochloride) 1% (w / v), Tris-HCl (pH 8.0) 50 mM, EGTA 0.1 mM, Tween 80 0.05%, Proclin 300 0.1%, rHSA 10 mg / mL, recombinant factor three protein 1.0 g. μg / mL, horseshoe crab tripeptide-pNA chromogenic substrate 0.5 mM, magnesium acetate 5 mM, polysorbate-20 0.01% (w / v), hydroxypropyl-β-cyclodextrin 0.5 mM; the unit of w / v is g / mL; II. Lyophilization to obtain recombinant horseshoe crab reagent lyophilized formulation Place the freeze-drying solution on the freeze dryer shelf and run the following program for freeze-drying: Cool down to -35℃ and keep warm for 3 hours; Cool to -50℃ and keep warm for 4 hours; Heat to -43℃ and hold for 11 hours; Heat to -36℃ and hold for 9 hours; Heat to -29℃ and hold for 7 hours; Heat to +25℃ and hold for 5 hours; Heat to +30℃ and hold for 3 hours; The vacuum level was controlled at 0.08–0.1 mbar throughout the process; After lyophilization, stopper and cap to obtain the recombinant horseshoe crab reagent lyophilized preparation for endotoxin detection.

[0032] Comparative Example 1: A recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection was prepared based on existing lyophilization formulations. The lyophilization solution was prepared according to patent CN108913707B. Specifically, sodium chloride, calcium chloride, magnesium chloride, and dextran were added to the recombinant three-factor protein mixture, horseshoe crab tripeptide-pNA chromogenic substrate, and reaction buffer. The mixture was then stirred and brought to a final volume of 100 mL. After sterile filtration through a 0.1 μm filter, the solution was dispensed into pyrogen-free vials (0.5 mL per vial) to obtain the lyophilized solution. The final concentrations of each component in the lyophilized solution were: sodium chloride 0.1% (w / v), calcium chloride 0.4 M, magnesium chloride 0.35 M, dextran 7.5% (w / v), recombinant three-factor protein 1.0 μg / mL, horseshoe crab tripeptide-pNA chromogenic substrate 0.5 mM, magnesium acetate 5 mM, polysorbate-20 0.01% (w / v), and hydroxypropyl-β-cyclodextrin 0.5 mM; where w / v is in g / mL. The freeze-drying process is the same as in Example 1.

[0033] Comparative Example 2: A recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection was prepared using an existing two-step lyophilization process. Using the freeze-drying solution prepared in step one of Example 1, the freeze-drying solution was placed on the shelf of the freeze dryer and freeze-dried using the traditional two-step freeze-drying process: Pre-freeze at -40℃ for 2 hours Sublimation at -20℃ for 18 hours Desorption and drying at 30℃ for 4 hours. The vacuum level was controlled at 0.08–0.1 mbar throughout the process; After freeze-drying, stopper and cap.

[0034] Comparative Example 3: Preparation of a recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection, lacking certain components. The lyophilized solution was prepared using Step 1 of Example 1. The final concentrations of each component in the lyophilized solution were as follows: mannitol 10% (w / v), sucrose 5% (w / v), sodium chloride 0.9% (w / v), Tris-HCl (pH 7.5) 50 mM, EGTA 0.5 mM, Tween 80 0.05% (w / v), Proclin 300 0.3% (w / v), recombinant factor three protein 1.0 μg / mL, chromogenic substrate 0.5 mM, magnesium acetate 5 mM, polysorbate-20 0.01% (w / v), hydroxypropyl-β-cyclodextrin 0.5 mM, without the addition of PVP K30, rHSA, ascorbic acid, inositol, and L-arginine; the unit of w / v is g / mL. The freeze-drying process is the same as in Example 1.

[0035] Experimental Example 1: Verification of Freeze-drying Performance The recombinant horseshoe crab reagent lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-3 were observed for appearance (color and collapse). 0.5 mL of pyrogen-free water was added to reconstitute the formulations, and the time required for complete dissolution was recorded. Subsequently, the reconstituted solutions were used to test a series of concentrations of bacterial endotoxin standards (0.005, 0.05, 0.5, 5 EU / ml) for performance verification experiments after lyophilization.

[0036] Table 1. Statistics on freeze-drying effects

[0037] Table 2 Performance verification after freeze-drying

[0038] The results are shown in Tables 1 and 2. The recombinant horseshoe crab reagent lyophilized formulation prepared in the embodiments of the present invention has a stable structure, rapid reconstitution, and is sensitive and linear at low concentrations (0.005 EU / mL). However, Comparative Examples 1 and 2 could not be detected at low concentrations, and the reaction time of Comparative Examples 1-3 was significantly prolonged at high concentrations, indicating that their activity was impaired.

[0039] Test Example 2: Long-term stability verification (stored at 2-8℃ for 12 months) The recombinant horseshoe crab reagent lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-3 were stored at 2-8℃ for 12 months, and then reconstituted with 0.5 mL of pyrogen-free water. The reconstituted solution was used to detect a series of bacterial endotoxin standards (0, 0.005, 0.05, 0.5, 5 EU / ml) to verify the performance and activity retention rate after lyophilization. The activity retention rate was calculated as follows: (activity after storage / initial activity) × 100% = (initial sample reaction time / sample reaction time after storage) × 100%. In this detection system, the activity was negatively correlated with the reaction time.

[0040] Table 3 Performance Verification After 12 Months of Storage

[0041] Table 4. Activity retention rate after 12 months of storage

[0042] The results, as shown in Tables 3 and 4, indicate that the recombinant horseshoe crab reagent lyophilized formulations prepared in Examples 1-3 of this invention maintained good detection capabilities against bacterial endotoxins at various concentrations after 12 months of storage, with activity retention rates all above 85% and a limit of detection of 0.005 EU / mL. In contrast, the recombinant horseshoe crab reagent lyophilized formulations prepared in Comparative Examples 1-3 were completely ineffective at low concentrations, and their activity loss exceeded 20% at high concentrations.

[0043] In summary, the recombinant horseshoe crab reagent lyophilized formulation and its gradient temperature-controlled lyophilization process provided by this invention significantly improve the physical stability and biological activity retention rate of the product, and have extremely high application value.

[0044] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A recombinant horseshoe crab reagent lyophilized formulation for bacterial endotoxin detection, characterized in that, The recombinant horseshoe crab reagent lyophilized formulation was obtained by lyophilizing a lyophilizing solution, which was prepared from the following components at the following final concentrations: Mannitol 5-10%, sucrose 2-5%, polyvinylpyrrolidone 1-3%, recombinant human serum albumin 5-10 mg / mL, ascorbic acid 0.1-0.5%, sodium chloride 0.8-1.0%, inositol 2-5%, L-arginine hydrochloride 1-2%, ethylene glycol diethyl ether diaminetetraacetic acid 0.1-0.5 mM, Tween 80 0.05-0.1%, Proclin 300 0.1-0.3%, Tris-HCl 50-100 mM (pH 7.0-8.0), recombinant horseshoe crab reagent 0.1-5.0 μg / mL, chromogenic substrate 0.1-1.0 mM, and reaction buffer.

2. The recombinant horseshoe crab reagent lyophilized formulation according to claim 1, characterized in that, The final concentrations of each component in the reaction buffer solution in the lyophilization solution are: magnesium acetate 1-10 mM, polysorbate-20 0.01-0.05%, and hydroxypropyl-β-cyclodextrin 0.1-1 mM.

3. The recombinant horseshoe crab reagent lyophilized formulation according to claim 1, characterized in that, The chromogenic substrate is a horseshoe crab tripeptide-pNA chromogenic substrate.

4. The recombinant horseshoe crab reagent lyophilized formulation according to claim 1, characterized in that, The final concentration of sodium chloride in the lyophilization solution is 0.9%.

5. The recombinant horseshoe crab reagent lyophilized formulation according to claim 1, characterized in that, The polyvinylpyrrolidone is PVP K30.

6. The recombinant horseshoe crab reagent lyophilized formulation according to claim 1, characterized in that, The preparation method of the lyophilization solution is as follows: Prepare a recombinant human serum albumin stock solution, filter and sterilize for later use; Weigh out mannitol, sucrose, polyvinylpyrrolidone, ascorbic acid, sodium chloride, inositol and L-arginine hydrochloride, dissolve in water, and add Tris-HCl, EGTA, Tween 80, Proclin 300 and the recombinant human serum albumin stock solution at pH 7.0-8.0; Then add recombinant horseshoe crab reagent, chromogenic substrate and reaction buffer; The mixture was brought to a final volume, mixed thoroughly, and then filtered to remove bacteria.

7. The method for preparing the lyophilized formulation of the recombinant horseshoe crab reagent according to any one of claims 1-6, characterized in that, Includes the following steps: The lyophilized liquid was dispensed into pyrogen-free containers and placed in a freeze dryer. The freeze-drying process was carried out under vacuum conditions controlled at 0.08 ~ 0.1 mbar according to the following procedure: Cool to -50~-20℃ and keep warm for 2-4 hours; Maintain at -50℃ for 3-5 hours; Heat to -45~-42℃ and hold for 10-12 hours; Heat to -40~-34℃ and keep warm for 8-10 hours; Heat to -35~-26℃ and keep warm for 6-8 hours; Heat to +25℃ and maintain the temperature for 4-6 hours; Heat to +30℃ and hold for 2-4 hours; After freeze-drying, stopper and cap the product to obtain the recombinant horseshoe crab reagent freeze-dried formulation.

8. The application of the recombinant horseshoe crab reagent lyophilized formulation according to any one of claims 1-6 in the detection of bacterial endotoxins.

9. The use of the recombinant horseshoe crab reagent lyophilized formulation according to any one of claims 1-6 in the preparation of reagents for bacterial endotoxin detection.

10. A kit for detecting bacterial endotoxins, characterized in that, The kit comprises the recombinant horseshoe crab reagent lyophilized formulation according to any one of claims 1-6.

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