A recombinant factor c protein protectant and uses thereof

Abstract

This invention discloses a recombinant factor C protein protectant and its application. Based on the final concentration of each component, the recombinant factor C protein protectant comprises the following components: 0.05-0.2% (w / v) ProClin 300, 100-300 mM sodium chloride, 1-10 mg / mL bovine serum albumin, 5-20% (w / v) trehalose, 10-50% (v / v) glycerol, 0.1-0.5% (w / v) Tween-20, and 0.1-1 mM pH 8.0 ethylene glycol diethyl ether diaminetetraacetic acid. This invention also discloses the application of the recombinant factor C protein protectant in the cold storage of recombinant factor C protein. The recombinant C factor protein protectant and the recombinant C factor protein are mixed to prevent the recombinant C factor protein from being affected by conditions such as aggregation, oxidation, freeze-thaw cycles, and metal ion-dependent inactivation, thereby improving the stability of the recombinant C factor protein during cold storage. This ensures that the recombinant C factor protein retains ≥90% of its activity after 12 months of cold storage, and maintains its accuracy and sensitivity in detecting endotoxins.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant factor C protein protectant, an endotoxin detection reagent containing the same, and their applications. Background Technology

[0002] Bacterial endotoxins are lipopolysaccharides found in the cell walls of Gram-negative bacteria. When bacteria die or undergo autolysis, they release endotoxins. If large amounts of endotoxins enter the bloodstream, they can cause a fever response, known as a "pyrogenic reaction." If endotoxins accumulate in the blood and exceed the body's clearance capacity, it can lead to varying degrees of endotoxemia. Therefore, all biological products, pharmaceuticals, and medical devices must pass bacterial endotoxin testing before use.

[0003] Limulus amebocyte lysate (LAL or Tachypleus amebocyte lysate, TAL) is the gold standard method for detecting endotoxins, extracted from the blood of the ancient marine animal, the horseshoe crab. Horseshoe crab blood contains a special hemocyanin called horseshoe cyanin, which specifically recognizes and binds to endotoxins. Once bound, it triggers a series of enzymatic reactions, ultimately leading to blood clotting. This unique biological reaction mechanism makes Limulus amebocyte lysate an ideal tool for detecting endotoxins in injectable drugs, biological products, vaccines, medical devices, and dialysis-related supplies, serving as a crucial guarantee for product safety in the pharmaceutical industry today.

[0004] With the increasing demand for endotoxin testing, the number of horseshoe crabs is gradually decreasing. The Chinese horseshoe crab and the round-tailed horseshoe crab were listed in China's "National Key Protected Wild Animals List" in February 2021, becoming Class II national key protected animals. Therefore, developing a rapid and accurate method for detecting endotoxins without using biological horseshoe crab blood has become particularly important.

[0005] Decades of research have confirmed that horseshoe crab blood contains factors C, B, G, procoagulant, and coagulogen. Endotoxins activate factor C, which in turn activates factor B. Activated factor B converts procoagulant into activated coagulase, which then cleaves coagulogen into coagulin, forming an insoluble gel. However, in horseshoe crab blood extracts, a cascade reaction also occurs, starting with factor G, reacting with (1,3)-β-D-glucan to induce coagulation. Therefore, to specifically detect endotoxins, it is necessary to remove factor G or inhibit the cascade reaction starting with factor G; otherwise, false positive results may occur in endotoxin detection.

[0006] Currently, the mainstream method for endotoxin detection uses horseshoe crab (Limulus amebocyte lysate) reagents prepared from horseshoe crab blood extracts. However, to protect the horseshoe crab family, ensure a stable supply of reagents, reduce batch-to-batch variability, and improve the specificity and stability of endotoxin detection, researchers have begun developing endotoxin detection reagents using artificially synthesized recombinant proteins. Studies have found that the recombinant C-factor method can replace traditional horseshoe crab reagents for endotoxin detection. The principle involves the recombinant C-factor directly cleaving a fluorescent substrate, and the resulting signal is recognized by a fluorescence microplate reader. This reaction process involves only one mechanism: utilizing the serine protease activity of C-factor after activation by endotoxin, the activated C-factor is added to the substrate. This substrate is characterized by a small peptide linked to a readable signal, such as an electrical or fluorescent signal. After cleavage by the protease, the signal is released and can be detected by the instrument. If the C-factor is not activated and does not possess serine protease activity, the signal will not be released, and no reading will be detected.

[0007] The 2021 edition of the Japanese Pharmacopoeia, J18, includes " <g4-4-180>The section on "Bacterial Endotoxin Testing and Alternative Methods for Detection of Endotoxins Using Recombinant Protein Reagents" includes recombinant factor C. The 2024 edition of the European Pharmacopoeia EP11.5 includes "2.6.32. Detection of Bacterial Endotoxins using Recombinant Factor C" as a separate chapter. The 2024 edition of the United States Pharmacopeia USP47... <86> This chapter details the technical specifications for the detection of bacterial endotoxins using recombinant Factor C. The final text of this chapter will be published in November 2024 and will officially take effect in May 2025. Furthermore, the "Application Guidelines for the 9251 Bacterial Endotoxin Test Method" in the General Technical Requirements of Part IV of the 2025 edition of the Chinese Pharmacopoeia includes "Appendix: Recombinant Factor C Method." The introduction of the recombinant Factor C method in these four pharmacopoeias demonstrates that the reliability of the recombinant Factor C methodology has gained global regulatory recognition. In terms of application scenarios, the recombinant Factor C method covers the entire pharmaceutical process (raw material testing, production process monitoring, and finished product release), and is particularly suitable for complex samples containing β-glucan (such as plant-derived biological products).

[0008] Recombinant factor C detection technology is gradually replacing traditional horseshoe crab reagents, but its industrialization urgently requires overcoming the technical barriers of protein stability. In the future, with innovation in protective agents and upgrades in production processes, recombinant factor C is expected to become the mainstream solution in the field of endotoxin detection, driving the pharmaceutical industry towards a sustainable, high-precision quality control system.

[0009] Despite the advantages of the recombinant factor C method, such as eco-friendliness (avoiding horseshoe crab trapping), high detection specificity (responding only to endotoxins), and high batch-to-batch consistency, protein stability issues have become a bottleneck for industrialization: physicochemical instability: susceptible to temperature fluctuations during liquid storage (disulfide bond breakage due to cold storage oxidation), stress-induced aggregation at the liquid-gas interface, and conformational inactivation due to repeated freeze-thaw cycles; risk of spontaneous activation: trace amounts of Ca... 2 + Metal ions may activate C factor precursors, causing background signal interference; Defects in the protection system: Existing solutions mostly use a single protectant (such as trehalose + BSA), which cannot simultaneously combat multiple inactivation pathways; Thimerosal preservatives are biotoxic, and the use of metal ion chelating agents alone exacerbates protein-metal ion imbalance precipitation.

[0010] Therefore, it is necessary to develop a protective agent for recombinant factor C protein to ensure its long-term stability under refrigeration conditions, thereby promoting the industrial production of recombinant factor C protein and the widespread adoption of recombinant factor C methods for detecting endotoxins. Summary of the Invention

[0011] To address the problems of the prior art, the first objective of this invention is to provide a recombinant factor C protein protectant that prevents the recombinant factor C protein from being affected by conditions such as aggregation, oxidation, freeze-thaw cycles, and metal ion-dependent inactivation, thereby improving the stability of the recombinant factor C protein during refrigeration, ensuring that the recombinant factor C protein retains ≥90% of its activity after 12 months of refrigerated storage, and maintaining its accuracy and sensitivity in detecting endotoxins.

[0012] The second objective of this invention is to provide a method for preparing the above-mentioned recombinant factor C protein protectant.

[0013] A third objective of this invention is to provide the application of the aforementioned recombinant factor C protein protectant in the cold storage of recombinant factor C protein.

[0014] The fourth objective of this invention is to compose an endotoxin detection kit using a recombinant factor C protein solution with added recombinant factor C protein protectant.

[0015] The fifth objective of this invention is to provide the application of the above-mentioned recombinant factor C protein protectant and the endotoxin detection kit containing it in endotoxin detection.

[0016] To achieve the above objectives, the present invention adopts the following technical solution.

[0017] In a first aspect, the present invention provides a recombinant factor C protein protectant, wherein, based on the final concentration of each component, the recombinant factor C protein protectant comprises the following components: 0.05-0.2% (w / v) ProClin 300, 100-300 mM sodium chloride (NaCl), 1-10 mg / mL bovine serum albumin (BSA), 5-20% (w / v) trehalose, 10-50% (v / v) glycerol, 0.1-0.5% (w / v) Tween-20, and 0.1-1 mM pH 8.0 ethylene glycol diethyl ether diaminetetraacetic acid (EGTA).

[0018] Furthermore, based on the final concentration of each component, the recombinant factor C protein protectant comprises the following components: 0.05% (w / v) ProClin 300, 200 mM sodium chloride, 1 mg / mL bovine serum albumin, 20% (w / v) trehalose, 10% (v / v) glycerol, 0.2% (w / v) Tween-20, and 1 mM pH 8.0 ethylene glycol diethyl ether diaminetetraacetic acid.

[0019] Furthermore, the endotoxin level of the recombinant C-protein protectant of the present invention should be below 0.005 EU / ml.

[0020] The protein protectant of the present invention, through the synergistic effect of a specific ratio of preservative (ProClin 300), salt solution (NaCl), protein stabilizer (BSA, trehalose, glycerol), surfactant (Tween-20) and metal ion chelating agent (EGTA), can maintain the conformation and enzyme activity of recombinant factor C protein for a long time under refrigeration conditions, avoiding protein denaturation caused by freezing or repeated freeze-thaw cycles. Specifically, ProClin 300 is used as a preservative, which can prevent microbial contamination at low concentrations while ensuring no inhibition of protein activity; NaCl maintains the ionic strength of the solution, stabilizes the charge distribution of proteins, and reduces protein aggregation caused by electrostatic repulsion; BSA inhibits protein surface adsorption and aggregation through molecular chaperone effects and steric hindrance; Trehalose forms a hydrophilic protective layer with glycerol and lowers the freezing point of the solution, maintaining the conformational integrity of proteins without freezing and completely eliminating protein aggregation or denaturation caused by repeated freeze-thaw cycles; Tween-20 reduces protein interfacial adsorption and aggregation by lowering the liquid-gas interfacial tension, while stabilizing the hydrophobic core region of the protein through hydrophobic interactions; EGTA chelates metal ions, blocking oxidation reactions and degradation of target proteins by metal-dependent proteases.

[0021] Secondly, this invention provides a method for preparing the above-mentioned recombinant factor C protein protectant. The method includes: dissolving all components in water according to the formulation concentration, mixing thoroughly, and then filtering to obtain the final product. Aseptic operation must be maintained during the preparation process, which is carried out entirely in a clean bench. According to a specific embodiment of this invention, the preparation process for 1L of the protein protectant includes the following steps: taking an appropriate volume of sterile water for injection, sequentially adding 0.05-0.2% (w / v) ProClin 300, 100-300mM NaCl, 1-10mg / mL BSA, 5-20% (w / v) trehalose, 10-50% (v / v) glycerol, 0.1-0.5% (w / v) Tween-20, and 0.1-1mM pH 8.0 EGTA, mixing thoroughly, and then adjusting the volume to 1L. The mixture is then filtered through a 0.1μm filter membrane, dispensed, and stored under cold (2-8℃).

[0022] Thirdly, the present invention provides the application of the above-mentioned recombinant factor C protein protectant in the cold storage of recombinant factor C protein.

[0023] The recombinant factor C protein protectant of the present invention is mainly used for the cold storage of recombinant factor C protein. Under normal circumstances, the cold storage temperature is 2-8°C and the cold storage time can be as long as at least 1 year.

[0024] Furthermore, the mass-to-volume ratio of recombinant factor C protein to recombinant factor C protein protectant is 0.02-0.2 mg: 1 mL.

[0025] Fourthly, the present invention provides an endotoxin detection kit, comprising a recombinant factor C protein solution with the above-mentioned recombinant factor C protein protectant added.

[0026] Fifthly, the present invention also claims protection for the above-mentioned recombinant factor C protein protectant and the use of the endotoxin detection kit thereon in endotoxin detection.

[0027] This invention utilizes a recombinant factor C protein solution with added recombinant factor C protein protectant, combined with an endotoxin detection reaction buffer and a fluorescent substrate, such as Boc-Val-Pro-Arg-AMC, to detect endotoxins in the product.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. Excellent long-term stability

[0030] Under refrigerated liquid storage conditions, the activity of recombinant factor C protein can be stably maintained for at least 1 year with an activity loss rate of less than 10%, which is significantly better than traditional storage methods (conventional refrigeration can only maintain activity for 1-2 weeks, while cryopreservation requires -80℃ equipment and carries the risk of freeze-thaw damage).

[0031] 2. Ease of operation and cost advantages

[0032] Direct liquid preservation eliminates cumbersome steps such as freezing, rehydration, and centrifugation, simplifying the production process and reducing equipment dependence (such as eliminating the need for ultra-low temperature freezers) and energy costs.

[0033] 3. Security and application compatibility

[0034] All components are biocompatible (with the non-toxic preservative ProClin 300 replacing traditional sodium azide), and can be directly used in diagnostic kits or biological products without additional purification steps; protects the reagents and ensures compatibility with endotoxin detection systems, avoids interference from buffer components with detection results, and improves the stability and reliability of the kit. Detailed Implementation

[0035] 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.

[0036] Unless otherwise specified, all reagents and consumables used in the following embodiments were purchased from conventional biochemical reagent manufacturers, and all experimental methods used were conventional methods in the art.

[0037] Example 1

[0038] Take an appropriate volume of sterile water for injection, and sequentially add 0.05% (w / v) ProClin 300, 300mM NaCl, 10mg / mL BSA, 5% (w / v) trehalose, 50% (w / v) glycerol, 0.1% (w / v) Tween-20, and 0.5mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1μm filter membrane to obtain recombinant factor C protein protectant 1. Add the above protein protectant 1 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 1.

[0039] Example 2

[0040] Take an appropriate volume of sterile water for injection, and sequentially add 0.05% (w / v) ProClin 300, 200mM NaCl, 1 mg / mL BSA, 20% (w / v) trehalose, 10% (v / v) glycerol, 0.2% (w / v) Tween-20, and 1mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1 μm filter membrane to obtain recombinant factor C protein protectant 2. Add the above protein protectant 2 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 2.

[0041] Example 3

[0042] Take an appropriate volume of sterile water for injection, and sequentially add 0.1% (w / v) ProClin 300, 100mM NaCl, 5mg / mL BSA, 10% (w / v) trehalose, 20% (w / v) glycerol, 0.5% (w / v) Tween-20, and 0.1mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1μm filter membrane to obtain recombinant factor C protein protectant 3. Add the above protein protectant 3 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 3.

[0043] Example 4

[0044] Take an appropriate volume of sterile water for injection, and sequentially add 0.2% (w / v) ProClin 300, 300mM NaCl, 1 mg / mL BSA, 5% (w / v) trehalose, 10% (w / v) glycerol, 0.2% (w / v) Tween-20, and 0.5mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1 μm filter membrane to obtain recombinant factor C protein protectant 4. Add the above protein protectant 4 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 4.

[0045] Example 5

[0046] Take an appropriate volume of sterile water for injection, and sequentially add 0.05% (w / v) ProClin 300, 100mM NaCl, 5mg / mL BSA, 20% (w / v) trehalose, 50% (v / v) glycerol, 0.1% (w / v) Tween-20, and 1mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1μm filter membrane to obtain recombinant factor C protein protectant 5. Add the above protein protectant 5 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 5.

[0047] Example 6

[0048] Take an appropriate volume of sterile water for injection and add, sequentially, 0.2% (w / v) ProClin 300, 200 mM NaCl, 10 mg / mL BSA, 10% (w / v) trehalose, 20% (w / v) glycerol, 0.5% (w / v) Tween-20, and 0.1 mM EGTA (pH 8.0) to a final concentration. After thorough dissolution and mixing, bring the volume to a final volume and filter through a 0.1 μm filter membrane to obtain recombinant factor C protein protectant 6. Add the above protein protectant 6 to the recombinant factor C protein and store at 2-8℃ to obtain recombinant factor C protein solution No. 6.

[0049] Example 7: Dilute the endotoxin standard, prepare the endotoxin reaction solution and plot the standard curve.

[0050] Take a disposable pyrogen-free glass tube, dissolve the endotoxin standard to 20 EU / ml, and then continue to perform serial dilutions (5, 0.5, 0.05, 0.005 EU / ml) to obtain endotoxin standard solutions of various concentrations. Add 100 μl of each concentration of endotoxin standard and endotoxin-free water to an endotoxin-free ELISA plate and preheat in a 37°C incubator. Then, mix the fluorescent substrate (Boc-Val-Pro-Arg-AMC), reaction buffer (100 mM Tris-Ac, pH 7.5, 50 mM NaCl), and the recombinant factor C protein solution with added protectant prepared in the above embodiments of the present invention in a 5:4:1 ratio to prepare the endotoxin reaction solution. After adding 100 μl of the reaction solution to the endotoxin standard or endotoxin-free water, immediately read the zero-point fluorescence value, and then place the ELISA plate in a 37°C incubator for one hour, and then read the value again using an ELISA reader. Subtract the zero-hour reading from the one-hour reading, then subtract the 0 EU / ml difference from the differences in readings of 5, 0.5, 0.05, and 0.005 EU / ml to obtain the final ΔRFU. Take the logarithm of this result and plot a standard curve, then calculate the correlation coefficient R. 2 The readings and linear differences of recombinant factor C protein solutions obtained after the addition of different protein protectants when detecting endotoxin reactions were compared.

[0051] Comparative Example 1

[0052] The recombinant factor C protein was diluted to the same concentration with sterile water for injection without adding any protein protectant, and then frozen at -20°C or below to obtain a control protein solution 1 without protectant stored at -20°C.

[0053] Comparative Example 2

[0054] The recombinant factor C protein was diluted to the same concentration with sterile water for injection without adding any protein protectant, and stored at 2-8°C to obtain a refrigerated control protein solution 2 without protectant.

[0055] Comparative Example 3

[0056] Take an appropriate volume of sterile water for injection and add ProClin 300 and 300mM NaCl to a final concentration of 0.05% (w / v) to obtain a contrast protein protectant. Add the contrast protein protectant to the recombinant factor C protein and store it at 2-8℃ to obtain contrast protein solution 3.

[0057] Comparative Example 4

[0058] Take an appropriate volume of sterile water for injection, and sequentially add 0.05% (w / v) ProClin 300, 300mM NaCl, 10mg / mL BSA, and 5% (w / v) trehalose to a final concentration. After thorough dissolution and mixing, bring the volume to a final depth and filter through a 0.1μm filter membrane to obtain the contrast protein protectant. Add the above contrast protein protectant to recombinant factor C protein and store at 2-8℃ to obtain contrast protein solution 4.

[0059] Comparative Example 5

[0060] Take an appropriate volume of sterile water for injection, and sequentially add 0.05% (w / v) ProClin 300, 300mM NaCl, 10mg / mL BSA, 5% (w / v) trehalose, 50% (w / v) glycerol, and 0.1% (w / v) Tween-20 to a final concentration. After thorough dissolution and mixing, bring the volume to a final depth and filter through a 0.1μm filter membrane to obtain the contrast protein protectant. Add the above protein protectant to recombinant factor C protein and store at 2-8℃ to obtain contrast protein solution 5.

[0061] Experiment 1: Effects of different protein protectants on the stability of recombinant factor C protein

[0062] The recombinant factor C protein solutions prepared in Examples 1-6 and Comparative Examples 1-5 were used for stability testing. The method described in Example 7 was followed, ensuring that the final concentration of recombinant factor C protein in the eleven groups was consistent at 20 μg / ml. The protein solutions prepared in Examples 1-6 and Comparative Examples 2-5 were subjected to accelerated temperature testing at room temperature (25°C) for two months. The sample from Comparative Example 1 was stored frozen at -20°C or below. The protective effects of different protective agents on the stability of recombinant factor C protein, and the influence of different protective agents on the measurement range and linearity of endotoxin response, are compared in Table 1.

[0063] Table 1. Effects of different protein protectants on the stability of recombinant factor C protein.

[0064] ΔRFU 0.005 EU / ml 0.05 EU / ml 0.5 EU / ml 5 EU / ml <![CDATA[R 2 ]]> room temperature acceleration Example 1 10.5 145 1279 9281 0.9958 room temperature acceleration Example 2 12.5 164 1715.5 11508.5 0.9956 room temperature acceleration Example 3 20 155 1508.5 10964.5 0.9994 room temperature acceleration Example 4 16.5 149 1406.5 10367.5 0.9994 room temperature acceleration Example 5 18.5 134 1163 9093.5 0.9997 room temperature acceleration Example 6 4.5 111 1124.5 8851 0.9891 -20℃ Comparative Example 1 44.5 199 2318.5 13851 0.9922 room temperature acceleration Comparative Example 2 -9 43 589 4516.5 / room temperature acceleration Comparative Example 3 8 67 638 5545.5 0.9999 room temperature acceleration Comparative Example 4 6 93 889.5 6948 0.9956 room temperature acceleration Comparative Example 5 6.5 103 1104.5 8485.5 0.9954

[0065] As shown in Table 1, the protective effects of different protein protectants added to recombinant factor C varied slightly in the examples, but all of them were higher than the readings of Comparative Example 2 without protectant or Comparative Examples 3-5 with some protectant added, indicating that the traditional protectant was not effective.

[0066] The sample in Example 2 had the highest residual reading, indicating that protein protectant 2 had the best protective effect on recombinant factor C protein. Combination 2 is the optimal choice: 0.05% (w / v) ProClin 300, 200mM NaCl, 1mg / mL BSA, 20% (w / v) trehalose, 10% (v / v) glycerol, 0.2% (w / v) Tween-20, 1mM EGTA, pH 8.0.

[0067] Experiment 2: Effects of different protein preservatives on the long-term cold storage stability of recombinant factor C protein

[0068] Recombinant factor C protein solutions were prepared using Examples 1-6 and Comparative Examples 1-5 for recombinant factor C protein stability testing. The method described in Example 7 was followed, ensuring that the final concentration of recombinant factor C protein in the eleven groups was consistent (20 μg / ml). The recombinant factor C protein solutions from Examples 1-6 and Comparative Examples 2-5 were stored at 2-8°C, while the protein solution from Comparative Example 1 was stored at -20°C or below. A one-year cold storage stability test was conducted. Following the method described in Example 7, the long-term cold storage protection effect of different protein preservatives on the stability of recombinant factor C protein, as well as their influence on the measurement range and linearity of endotoxin response, were tested, as shown in Table 2.

[0069] Table 2. Effects of different protein protectants on the long-term cold storage stability of recombinant factor C protein.

[0070] ΔRFU 0.005 EU / ml 0.05 EU / ml 0.5 EU / ml 5 EU / ml stability <![CDATA[R 2 ]]> refrigeration Example 1 33 306 2255 12411 92％ 0.9965 refrigeration Example 2 15.5 211.5 1869 12743.5 95％ 0.9951 refrigeration Example 3 8 185.5 1921.5 12561 93％ 0.9865 refrigeration Example 4 30 267.5 2184 12519 93％ 0.9974 refrigeration Example 5 15 160 1664 12151.5 90％ 0.9984 refrigeration Example 6 19 187 1772.5 12216 91％ 0.9985 -20℃ Comparative Example 1 20 226.5 2127 13471 100％ 0.9964 refrigeration Comparative Example 2 1 49 417 6129 45％ 0.9846 refrigeration Comparative Example 3 23.5 173.5 1332 7024.5 52％ 0.998 refrigeration Comparative Example 4 15 171 1329 8774.5 65％ 0.9966 refrigeration Comparative Example 5 4.5 130 1307 9767.5 73％ 0.9851

[0071] As shown in Table 2, the protein protectants in the examples showed excellent protective effects on recombinant factor C protein after one year of refrigerated storage. The protein activity after storage was ≥90% (compared to the initial activity), which was much higher than the protective effects of no protectant or traditional protectants (comparative examples 2-5).

[0072] Experimental Example 3: Detection of the accuracy and anti-interference ability of measuring endotoxin content in sodium chloride injection stock solution after adding various protein protectants to recombinant factor C protein.

[0073] The recombinant factor C protein solutions prepared in Examples 1-6 and Comparative Examples 1-2 were refrigerated and stored for 12 months. Endotoxin reaction experiments were performed according to the method described in Example 7 to detect the endotoxin content of the undiluted sodium chloride injection solution. Spiking experiments were performed on the samples, and the results were compared with those obtained by the traditional Limulus amebocyte lysate (LAL) reagent dynamic turbidimetric method (Table 3).

[0074] Table 3. Results of measuring endotoxin content in sodium chloride injection stock solution in Examples 1-6 and Comparative Examples 1-2.

[0075]

[0076] As shown in Table 3, the absence of a preservative under long-term refrigeration conditions leads to a decrease in the anti-interference ability of recombinant factor C protein in detecting endotoxin reactions, while the recombinant factor C protein solutions prepared in Examples 1-6 exhibit excellent anti-interference ability.

[0077] Example 4: Detection of the accuracy and anti-interference ability of the recombinant factor C protein solution containing the protective agent of the present invention in measuring the endotoxin content of various samples.

[0078] The recombinant factor C protein solution containing the protective agent prepared in Example 2 was used to conduct endotoxin reaction experiments according to the method described in Example 7, and the endotoxin content of several injected samples was detected. Spiking experiments were performed on the samples, and the results were compared with those of the traditional Limulus amebocyte lysate (LAL) reagent dynamic turbidimetric method, as shown in Table 4.

[0079] Table 4. Results of measuring endotoxin content in different injection samples using the recombinant factor C protein solution prepared in Example 2.

[0080]

[0081] As shown in Table 4, the protein protectant of the present invention has no inhibitory effect on the accuracy and sensitivity of the recombinant factor C protein in detecting endotoxin reactions, has excellent anti-interference ability, and can accurately measure various injection samples.

[0082] The endotoxin detection reagents prepared in other embodiments also have the same excellent performance, and will not be listed one by one here.

[0083] 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 factor C protein protectant, characterized in that, Based on the final concentration of each component, the recombinant factor C protein protectant consists of the following components: 0.05-0.2% (w / v) ProClin 300, 100-300 mM sodium chloride, 1-10 mg / mL bovine serum albumin, 5-20% (w / v) trehalose, 10-50% (v / v) glycerol, 0.1-0.5% (w / v) Tween-20, and 0.1-1 mM pH8.0 ethylene glycol diethyl ether diaminetetraacetic acid.

2. The recombinant factor C protein protectant according to claim 1, characterized in that, Based on the final concentration of each component, the recombinant factor C protein protectant consists of the following components: 0.05% (w / v) ProClin 300, 200 mM sodium chloride, 1 mg / mL bovine serum albumin, 20% (w / v) trehalose, 10% (v / v) glycerol, 0.2% (w / v) Tween-20, and 1 mM pH 8.0 ethylene glycol diethyl ether diaminetetraacetic acid.

3. The recombinant factor C protein protectant according to claim 1, characterized in that, The endotoxin level of the recombinant factor C protein protectant is below 0.005 EU / ml.

4. The method for preparing the recombinant factor C protein protectant according to any one of claims 1-3, characterized in that, The preparation method includes: dissolving all components in water according to the formula concentration, mixing thoroughly, and then filtering to obtain the final product.

5. The preparation method according to claim 4, characterized in that, Filtration was performed using a 0.1 μm filter membrane.

6. The use of the recombinant factor C protein protectant according to any one of claims 1-3 in the cold storage of recombinant factor C protein.

7. The application according to claim 6, characterized in that, The refrigeration temperature is 2-8℃, and the refrigeration time is at least 1 year.

8. The application according to claim 6, characterized in that, The mass-to-volume ratio of recombinant factor C protein to recombinant factor C protein protectant is 0.02-0.2 mg : 1 mL.

9. An endotoxin detection kit, characterized in that, This includes a recombinant factor C protein solution containing the recombinant factor C protein protectant as described in any one of claims 1-3.

10. The application of the recombinant factor C protein protectant according to any one of claims 1-3, or the endotoxin detection kit according to claim 9, in endotoxin detection, wherein the application is a non-diagnostic application.

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