Bacterial endotoxin test method for disposable liquid storage bag

Abstract

The invention relates to the technical field of biological detection, in particular to a bacterial endotoxin detection method for a disposable liquid storage bag. The bacterial endotoxin test method of the disposable liquid storage bag comprises the following steps: filling water into the disposable liquid storage bag, and then carrying out constant-temperature shaking table treatment to prepare a test solution; performing an interference preliminary screening test on the test solution to determine the minimum non-interference dilution multiple of the test solution; and diluting the test solution by selecting the minimum non-interference dilution multiple determined by the interference preliminary screening test, and then detecting the bacterial endotoxin by adopting a dynamic color development method. According to the bacterial endotoxin detection method for the disposable liquid storage bag, the consumption of adopted key reagents is low, and the cost is remarkably reduced; a matched full-automatic system can detect as many as 19 samples at a time, the detection period is greatly shortened, and the error rate of manual operation is reduced.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for testing bacterial endotoxins using disposable storage bags. Background Technology

[0002] In the modern biopharmaceutical field, single-use systems have been widely adopted as a novel process equipment solution. These systems encompass various types of consumables, such as single-use storage bags, sampling bags, bioreactors, stirring systems, and mixing systems. Due to their significant advantages, including high cost-effectiveness, ease of operation, excellent sterility, and effective prevention of cross-contamination, single-use technology plays a crucial role in all stages of the biopharmaceutical process.

[0003] Disposable storage bags are among the most widely used components, serving multiple critical applications such as the storage and transport of buffers and culture media, the storage and transfer of process intermediates, the collection of purified components, and the storage of bulk solutions and final products. With their widespread use, the industry's quality control standards for disposable storage bags have become increasingly mature and stringent. Bacterial endotoxins are a key indicator of their quality. Bacterial endotoxins are lipopolysaccharide complexes on the outer wall of Gram-negative bacterial cell walls; even at extremely low concentrations, they can induce severe immune responses in the host, such as fever. Therefore, strict control is essential during pharmaceutical manufacturing. Many pharmaceutical companies not only require suppliers' products to meet industry-standard levels (e.g., <0.25 EU / mL) but also propose even stricter internal control standards (e.g., <0.125 EU / mL), and desire quantitative detection and continuous trend analysis of bacterial endotoxin levels in products to promptly identify potential risks during production.

[0004] Currently, the traditional method for detecting bacterial endotoxins in the pharmaceutical industry is the gel permeabilization test (LAL). However, this method has a number of drawbacks, making it increasingly difficult to meet the needs of modern pharmaceutical industry development. The main problems include: (1) Limitations of the test results: The gel electrophoresis method is essentially a limit test or semi-quantitative detection method. It can only determine whether the endotoxin content in the sample exceeds a certain threshold (qualified or unqualified), but cannot provide a precise and quantifiable concentration value. This makes it impossible for manufacturers to conduct accurate trend analysis and risk warning of product quality.

[0005] (2) Resource consumption and environmental protection issues: The traditional gelation method requires a large amount of horseshoe crab reagent. The source of horseshoe crab reagent is the marine organism horseshoe crab. Since it has been listed as a protected animal, the large-scale consumption of horseshoe crab resources does not meet the policy requirements of sustainable development and environmental protection.

[0006] (3) Data integrity risk: The gel electrophoresis detection process involves a large number of manual operations, such as dilution, sample addition and result interpretation, which are not only prone to operational errors, but also difficult to meet increasingly stringent data integrity requirements. The detection process lacks automated monitoring, which is easily challenged in regulatory audits.

[0007] In summary, the gel electrophoresis method widely used in existing technologies has significant shortcomings in terms of quantitative capability, resource conservation, and data reliability, failing to meet the needs of the biopharmaceutical industry for accurate, efficient, and reliable quality control of bacterial endotoxins in products such as disposable storage bags. Therefore, there is an urgent need to develop a novel detection method that can overcome these deficiencies. Summary of the Invention

[0008] This invention provides a method for testing bacterial endotoxins using disposable storage bags, in order to solve the aforementioned problems existing in the prior art.

[0009] This invention provides a method for testing bacterial endotoxins using a disposable storage bag, comprising: filling the disposable storage bag with water and then treating it in a constant temperature shaker to prepare a test solution; performing an interference screening test on the test solution to determine the minimum non-interference dilution factor; diluting the test solution using the minimum non-interference dilution factor determined by the interference screening test, and then performing bacterial endotoxin detection using a dynamic colorimetric method.

[0010] This invention employs a specific method to extract bacterial endotoxins from disposable storage bags, ensuring accurate and efficient extraction. It also introduces interference testing and dynamic colorimetric methods to construct a complete and scientific quantitative detection framework. This not only significantly improves the accuracy and reliability of the detection results but also provides precise data support for product quality trend analysis and risk warning, thereby achieving efficient, accurate, and reliable quality monitoring of bacterial endotoxin levels in disposable storage bag products.

[0011] According to the method for testing bacterial endotoxins in disposable liquid storage bags of the present invention, the ratio of the inner surface area of ​​the disposable liquid storage bag to the volume of added water is (5-7):1, wherein the unit of inner surface area is cm². 2 The unit for volume is mL; the unit for internal surface area is m². 2 The volume is measured in liters (L). This precise ratio ensures efficient transfer of bacterial endotoxins from the surface of the disposable storage bag into the extract during the extraction process. It effectively avoids two extreme situations: excessive dilution of the target substance due to excessive liquid volume, resulting in a false negative (low detection limit), and insufficient extraction due to insufficient liquid volume, leading to incomplete extraction (low results).

[0012] According to the method for bacterial endotoxin testing using a disposable storage bag as described in this invention, during the pretreatment of the test sample, the temperature of the constant temperature shaker is 36-38℃, the shaking rate is 40-60 rpm, and the extraction time is ≥1h (preferably 1-2h).

[0013] In this invention, the temperature of the constant-temperature shaker (36-38℃) simulates a temperature close to that of the human body, which is the optimal temperature range for the Limulus amebocyte lysate (LAL) reagent reaction, while also gently promoting the dissolution of endotoxin molecules. The shaking rate (40-60 rpm) ensures sufficient and uniform contact between the extract and the inner surface of the storage bag, while avoiding excessive degradation of the bag material due to violent shaking, which could release more interfering substances. The specified minimum extraction time (≥1 h) ensures that the endotoxin has sufficient time to transfer from the surface to the extract. These parameters together constitute a controlled and efficient extraction condition, maximizing the recovery of endotoxins while minimizing the introduction of interfering substances, providing a high-quality test solution for subsequent accurate quantitative detection.

[0014] The method for testing bacterial endotoxins in disposable storage bags according to the present invention includes the following steps: S1. Reliability test of standard curve: Prepare standard bacterial endotoxin solutions with at least three concentration gradients, and use the standard bacterial endotoxin solutions to conduct a standard curve reliability test to determine the detection standard curve of the dynamic colorimetric method. S2. Prepare the sample solution: Water is added to a disposable storage bag and then subjected to constant temperature shaking to prepare a test solution; the test solution is diluted to no more than the maximum effective dilution factor to prepare test solutions and spiked test solutions at various dilution factors; Prepare standard bacterial endotoxin solutions with at least three concentration gradients as standard solutions; Water used for bacterial endotoxin testing was used as a negative control. S3. Interference screening test: S31: Inject the negative control, standard solution, test solution at each dilution factor, and spiked test solution at each dilution factor into the fully automated bacterial endotoxin testing system in the following order, with at least two parallel tubes for each sample; add 20-30 µL of Limulus amebocyte lysate (LAL) reagent to each well to complete the preparation of the ELISA plate; incubate the prepared ELISA plate in the ELISA reader, and then collect data; S32: Perform regression analysis on the collected data to determine the validity of the experiment and determine the minimum non-interference dilution factor of the test solution; S4. Bacterial endotoxin detection: After diluting the test solution with the minimum non-interference dilution factor confirmed by the interference screening test, perform bacterial endotoxin detection according to steps S31 and S32.

[0015] This invention constructs a systematic, quantitative, and validated detection system, overcoming the shortcomings of existing technologies (such as gel electrophoresis) which can only perform limit or semi-quantitative detection and cannot accurately assess product quality or conduct trend analysis. Step S1 establishes a reliable standard curve, ensuring the foundation of quantitative detection, namely accuracy and linearity. Step S3 identifies and determines the conditions (i.e., the minimum non-interfering dilution factor) for eliminating or reducing the influence of possible interfering substances in the extract of the storage bag through systematic interference screening, ensuring the authenticity of the detection results. Step S4 applies the aforementioned validated conditions for final detection. The testing method of this invention can achieve accurate and reliable quantitative determination of bacterial endotoxin levels in disposable storage bags, providing precise data support for product quality control, thereby enabling quality trend analysis and risk warning, and significantly improving the monitoring level and reliability of product quality.

[0016] According to the method for testing bacterial endotoxins in disposable storage bags of the present invention, the fully automated bacterial endotoxin testing system operates at a wavelength of 340-660 nm, a testing temperature of 36-38 °C, and a sampling time of 60-120 min.

[0017] This invention standardizes these key instrument parameters, eliminating variations caused by differences in instrument settings, ensuring the quality of reaction kinetic data, and achieving high-precision quantitative analysis. Specifically, the operating wavelength of 340-660 nm covers the maximum absorption wavelength (preferably 405 nm) of the Limulus amebocyte lysate (LAL) reagent reaction products in the dynamic colorimetric method, ensuring sensitive signal detection; the testing temperature of 36-38℃ is the optimal activity temperature for the enzymatic reaction in the LAL reagent, ensuring a fast and stable reaction rate; and the sampling time of 60-120 min allows for complete capture of the kinetic reaction curve.

[0018] According to the method for testing bacterial endotoxins using a disposable storage bag as described in this invention, in step S2, the maximum effective dilution factor of the test solution is 6.25.

[0019] In this invention, the dilution operations performed to overcome interference must not exceed this multiple; otherwise, endotoxins below the limit may be over-diluted and undetectable (i.e., false negative results).

[0020] According to the method for testing bacterial endotoxins using a disposable storage bag as described in this invention, in step S2, the dilution factor of the test solution is 1-4 times.

[0021] This invention limits the screening to this range, which can avoid unnecessary high-dilution tests, thereby saving reagents, consumables and time, and making the interference test process simpler and more efficient.

[0022] According to the method for testing bacterial endotoxins using a disposable storage bag as described in this invention, the minimum non-interfering dilution factor of the test solution is 1.

[0023] In this invention, the minimum non-interfering dilution factor is 1, meaning that the recovery rate of the test solution is already within an acceptable range (50%-200%) without any dilution. This indicates that the storage bag material itself and the extraction process introduce very few interfering substances. In routine quality control, the undiluted solution can be used directly for testing, avoiding errors that may be introduced by the dilution step, while also enabling the method to achieve a theoretically excellent detection limit.

[0024] According to the method for bacterial endotoxin testing using a disposable storage bag as described in this invention, the preparation method of the horseshoe crab reagent is to add 0.3-0.4 volume parts of the reconstituted lyophilized horseshoe crab powder to 0.3-0.4 volume parts of water for bacterial endotoxin testing and dissolve it.

[0025] According to the method for testing bacterial endotoxins using a disposable storage bag as described in this invention, in step S2, the concentration of the standard solution is 0.02-2 EU / mL. The lower limit of this concentration range (0.02 EU / mL) matches the sensitivity of the Limulus amebocyte lysate (LAL) reagent, forming the basis for accurate quantification; the upper limit (2 EU / mL) is significantly higher than the product's internal control limit (e.g., 0.125 EU / mL), providing a sufficient measurement range to address potential contamination events.

[0026] According to the method for testing bacterial endotoxins using disposable storage bags of the present invention, in step S32, the criteria for judging the validity of the experiment are: the correlation coefficient of the standard curve |r| ≥ 0.980, the recovery rate of the test solution is between 50% and 200%, the reaction time of the negative control is greater than the reaction time at the lowest point of the standard curve, and the CV of the result per parallel tube is ≤ 20%.

[0027] The beneficial effects of this invention are: The bacterial endotoxin testing method using disposable storage bags provided by this invention employs a micro-volume dynamic colorimetric method, which consumes only one-tenth of the key reagents required by the traditional gel electrophoresis method, significantly reducing costs. Simultaneously, the accompanying fully automated system can simultaneously test up to 19 samples, greatly shortening the testing cycle and reducing the error rate caused by human operation. This invention utilizes higher sensitivity and a wider detection range to ensure accurate measured values, and by scientifically evaluating and selecting appropriate dilution factors to overcome sample interference, it ensures the authenticity of the results and fully meets regulatory requirements. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] The raw materials used in the following experimental examples and embodiments are from the following sources: Test sample: Disposable liquid storage bags, batch numbers H20250620BM1115E01, H20250621BM1115E01, H20250622BM1115E01, source: Bailinke Pharmaceutical Equipment Technology (Jiangsu) Co., Ltd.; Limulus amebocyte lysate (LAL) reagent: Manufacturer: Zhanjiang Andus Biotechnology Co., Ltd., Specification: 0.35mL / vial, Detection range: 10~0.01EU / mL; Bacterial endotoxin: Manufacturer: Zhanjiang Andus Biotechnology Co., Ltd., Specification: 11 EU / vial.

[0030] Water for bacterial endotoxin testing: Manufacturer: Zhanjiang Andus Biotechnology Co., Ltd., endotoxin level less than 0.003 EU / mL; Fully Automated Bacterial Endotoxin Testing System: Manufacturer: Zhanjiang Andus Biotechnology Co., Ltd., Model: Robot 100.

[0031] Example 1 This embodiment provides a method for testing bacterial endotoxins using disposable storage bags, including the following steps: 1. Reliability test of standard curve 1.1 Take one vial of 11 EU / vial of reconstituted bacterial endotoxin working standard, add 1.1 mL of water for bacterial endotoxin testing, dissolve thoroughly and vortex for 5 min to obtain a bacterial endotoxin solution with a concentration of 10 EU / mL, and transfer to a reagent tube. Set the concentrations of the bacterial endotoxin standard to 2 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, and dilute stepwise, mixing thoroughly. Prepare at least three parallel tubes for each concentration, and also prepare two negative controls.

[0032] 1.2 Preparation of Limulus Amebocyte Lysate (LAL) Reagent: Take 0.35 mL of the reconstituted lyophilized powder, add 0.35 mL of water for bacterial endotoxin testing, let stand until dissolved and clear, transfer and combine into one ampoule for later use.

[0033] 1.3 When the reaction time of the negative control is greater than the reaction time at the lowest point of the standard curve, all data are subjected to linear regression analysis. According to the linear regression analysis, the absolute value of the correlation coefficient (r) of the standard curve should be greater than or equal to 0.980 for the experiment to be valid and the reliability test of the standard curve to meet the requirements.

[0034] 2. Prepare the sample solution: 2.1 Pretreatment of the test sample Place the disposable liquid storage bag in a sterilized and purified laminar flow hood, based on the sample's internal surface area (1246 cm²). 2 The ratio of the volume of water in the graduated cylinder to the volume of the extractant (mL) is 6:1. Add 208 mL of the calculated volume of ultrapure water to the graduated cylinder. In a clean bench, remove the plug from the sample bag and pour the water from the graduated cylinder into the sample bag through the interface. After adding the ultrapure water, tighten the stopcock. Place the sample bag in a constant-temperature shaker, set the temperature to 37±1℃, and the shaking speed to 50 rpm. Perform dynamic extraction in the shaker for at least 1 hour. After extraction, remove the sample bag and place it in a clean bench. Open the stopcock and pour approximately 3 mL of the extract into a test tube. Mix thoroughly to obtain the test solution.

[0035] 2.2 Sequence Setup Table 1 serial number Endotoxin concentration Solution with added endotoxin Number of parallel tubes A none Test solution At least 2 B The concentration at the midpoint (or adjacent point) of the standard curve is set as λm. Test solution At least 2 C At least 3 concentrations (the lowest point is set as λ). Check water usage At least 2 per concentration D none Check water usage At least 2 2.3 Selection of Dilution Factor One batch of samples was selected for dilution factor investigation. The maximum dilution factor (MVD) was calculated to be 6.25 times. A standard curve was established using the confirmed standard curve concentration points of 2 EU / mL, 0.2 EU / mL, and 0.02 EU / mL. Dilution factors not exceeding MVD were selected to prepare samples and spiked solutions at 1X, 2X, and 4X dilutions. Two parallel wells were prepared for interference screening tests, and the recovery rate was calculated. The recovery rate should meet the requirement of 50%-200%.

[0036] MVD=cL / λ=1.0ml / ml*0.125EU / ml÷0.02EU / ml=6.25 (times) Interference screening tests were conducted using bacterial endotoxin standards and horseshoe crab reagents from two different manufacturers (Zhanjiang Andus and Xiamen Horseshoe Crab Biotechnology) to confirm whether the screening test results from different manufacturers were consistent and whether the recovery rates met the requirements.

[0037] 3. Interference screening test 3.1 Experimental Procedure 3.1.1 Preparation of Zhanjiang Andus Standard Curve: Take one ampoule of 11 EU / vial of reconstituted bacterial endotoxin working standard, add 1.1 mL of bacterial endotoxin test water, dissolve thoroughly and vortex for 5 min to obtain a bacterial endotoxin solution with a concentration of 10 EU / mL, and transfer to a reagent tube. Set the concentration of bacterial endotoxin standard to 2 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, and dilute stepwise, mixing thoroughly. Preparation of Limulus Amebocyte Lysate (LAL) Reagent: Take 0.35 mL / vial of reconstituted lyophilized powder, add 0.35 mL of bacterial endotoxin test water, let stand to dissolve until clear, transfer and combine into one ampoule for later use.

[0038] Instrument parameters: Working wavelength: 405nm; Instrument temperature: 37℃; OD value: 0.1; Test method: Dynamic color development; Sampling time: 60min.

[0039] 3.1.2 Preparation of Standard Curve for Limulus Amebocyte Lysate (LIL) in Xiamen: Take one vial of 100 EU / vial of reconstituted bacterial endotoxin working standard, add 2 mL of water for bacterial endotoxin testing, and vortex mix thoroughly for 15 min to obtain a bacterial endotoxin solution with a concentration of 50 EU / mL. Set the concentration of the bacterial endotoxin standard to 2 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, and dilute stepwise, mixing thoroughly each time. Preparation of Limulus Amebocyte Lysate (LIL) Reagent: Take 0.5 mL / vial of reconstituted lyophilized LIL reagent powder, add 0.5 mL of LIL reagent dissolving solution, allow to stand until clear, and transfer to a single vial for later use.

[0040] Instrument parameters settings: Working wavelength: 405nm; Instrument temperature: 37℃; OD value: set according to the value recommended in the manufacturer's report; Test method: dynamic color development; Sampling time: 120min.

[0041] 3.1.3 Sequence establishment and ELISA plate preparation: Samples are injected in sequence. The fully automated bacterial endotoxin testing system automatically transfers in 2 negative controls (water for bacterial endotoxin testing), 3 standards (concentration points from low to high: 0.02, 0.2, 2, with 2 parallel samples for each point), 2 parallel samples of test samples (1X, 2X, 4X), and 2 parallel samples of spiked test samples (1X, 2X, 4X). 25µL of Limulus Amebocyte Lysate (LAL) reagent is added to each well to complete the ELISA plate preparation.

[0042] 3.1.4 Place the prepared ELISA plate into the ELISA reader for incubation. After the data collection time is set, the experiment is complete.

[0043] 3.2 Analyze and confirm the collected data. 3.2.1 System Applicability Confirmation Negative control confirmation: If the reaction time of the negative control is greater than the reaction time at the lowest point of the standard curve, the negative control is valid.

[0044] Standard curve confirmation: correlation coefficient |r|≥0.980, CV value of parallel 2 holes≤20%, standard curve is valid.

[0045] For the test solution: the CV value of two parallel wells ≤20% and the recovery rate R (%) in the range of 50%-200% are valid; otherwise, the results are invalid.

[0046] 3.2.2 Result Judgment Comparing the interference screening test results of Zhanjiang Andus and Xiamen Horseshoe Crab Biotechnology, the recovery rate of the test samples should meet the requirements of 50%-200% at the same dilution factor, and there should be no significant difference in interference.

[0047] Table 2 Results of initial interference screening test

[0048] 4. Interference Experiment 4.1 Selection of Dilution Factor Three batches of samples were selected, and reagents from Zhanjiang Andus and Xiamen Houshengke were used. The dilution factor (1X) confirmed by the interference screening test was selected to dilute the test samples. The test samples and test sample spiked solutions were prepared in parallel in two wells to compare whether the recovery rates of the three batches of test samples all met the requirements.

[0049] 4.2 Experimental Procedure The experimental procedure is consistent with that in 3.1.

[0050] 4.3 System Suitability Confirmation Consistent with the system suitability confirmation in 3.2.1.

[0051] 4.4 Result Confirmation The recovery rate of three batches of test samples tested using reagents from two different manufacturers should meet the requirement of 50%-200% at the selected dilution factor. This confirms the differences in the results of testing three batches of test samples using reagents from the same manufacturer and the consistency of the reaction of this product with reagents from different manufacturers.

[0052] Table 3 Interference Test Results

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for testing bacterial endotoxins in disposable storage bags, characterized in that, include: The test solution is prepared by filling a disposable liquid storage bag with water and then treating it on a constant temperature shaker. An interference screening test was performed on the test solution to determine the minimum non-interference dilution factor. The test solution was then diluted with the minimum non-interference dilution factor determined by the interference screening test, and bacterial endotoxins were detected using a dynamic colorimetric method.

2. The method for testing bacterial endotoxins in disposable liquid storage bags according to claim 1, characterized in that, The ratio of the inner surface area of ​​the disposable liquid storage bag to the volume of added water is (5-7):1, where the unit of inner surface area is cm². 2 The unit for volume is mL; the unit for internal surface area is m². 2 The unit of volume is L; And / or, the temperature of the constant temperature shaker is 36-38℃, the shaking rate is 40-60rpm, and the extraction time is ≥1h.

3. The method for testing bacterial endotoxins in disposable storage bags according to claim 1 or 2, characterized in that, The minimum non-interfering dilution factor for the test solution is 1.

4. The method for testing bacterial endotoxins in disposable storage bags according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Reliability test of standard curve: Prepare standard bacterial endotoxin solutions with at least three concentration gradients, and use the standard bacterial endotoxin solutions to conduct a standard curve reliability test to determine the detection standard curve of the dynamic colorimetric method. S2. Prepare the sample solution: Water is added to a disposable storage bag and then subjected to constant temperature shaking to prepare a test solution; the test solution is diluted to no more than the maximum effective dilution factor to prepare test solutions and spiked test solutions at various dilution factors; Prepare standard bacterial endotoxin solutions with at least three concentration gradients as standard solutions; Water used for bacterial endotoxin testing was used as a negative control. S3. Interference screening test: S31: Inject the negative control, standard solution, test solution at each dilution factor, and spiked test solution at each dilution factor into the fully automated bacterial endotoxin testing system in the following order, with at least two parallel tubes for each sample; add 20-30 µL of Limulus amebocyte lysate (LAL) reagent to each well to complete the preparation of the ELISA plate; incubate the prepared ELISA plate in the ELISA reader, and then collect data; S32: Perform regression analysis on the collected data to determine the validity of the experiment and determine the minimum non-interference dilution factor of the test solution; S4. Bacterial endotoxin detection: After diluting the test solution with the minimum non-interference dilution factor confirmed by the interference screening test, perform bacterial endotoxin detection according to steps S31 and S32.

5. The method for testing bacterial endotoxins in disposable storage bags according to claim 4, characterized in that, The fully automated bacterial endotoxin testing system operates at a wavelength of 340-660nm, a testing temperature of 36-38℃, and a sampling time of 60-120min.

6. The method for testing bacterial endotoxins in disposable storage bags according to claim 4 or 5, characterized in that, In step S2, the maximum effective dilution factor of the test solution is 6.

25.

7. The method for testing bacterial endotoxins in disposable storage bags according to any one of claims 4-6, characterized in that, In step S2, the dilution factor of the test solution is 1-4 times.

8. The method for testing bacterial endotoxins in disposable storage bags according to any one of claims 4-7, characterized in that, In step S31, the method for preparing the horseshoe crab reagent is to add 0.3-0.4 volume parts of the reconstituted lyophilized horseshoe crab powder to 0.3-0.4 volume parts of water for bacterial endotoxin testing and dissolve it.

9. The method for testing bacterial endotoxins in disposable storage bags according to any one of claims 4-8, characterized in that, In step S2, the concentration of the standard solution is 0.02-2 EU / mL.

10. The method for testing bacterial endotoxins in disposable storage bags according to any one of claims 4-9, characterized in that, In step S32, the criteria for judging the validity of the experiment are: the correlation coefficient of the standard curve |r| ≥ 0.980, the recovery rate of the test solution is between 50% and 200%, the reaction time of the negative control is greater than the reaction time at the lowest point of the standard curve, and the CV of the result per parallel tube is ≤ 20%.

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