A pre-treatment kit and pre-treatment method for detecting endotoxin in a biological product sample

Abstract

The application provides a pre-treatment kit and a pre-treatment method for endotoxin detection samples of biological products, and the pre-treatment kit for endotoxin detection samples of nuclease biological products is characterized in that the pre-treatment kit comprises a lysing agent A, a neutralizing agent B and a stabilizing agent C; the lysing agent A is a protein lysing enzyme, the neutralizing agent B is a hydrogen peroxide solution, and the stabilizing agent C is a magnesium sulfate solution. The pre-treatment kit and the pre-treatment method can be applied to the pre-treatment of various endotoxin detection methods such as a gel method, a chromogenic method and a recombinant C factor method, so that the recovery rate of various nuclease biological products reaches the standard.

Description

Technical Field

[0001] This invention relates to a pretreatment kit for endotoxin detection samples of biological products, and also to a pretreatment method for endotoxin detection samples of biological products, belonging to the field of biological product detection. Background Technology

[0002] Over the past decade, the manufacturing of biopharmaceuticals has experienced explosive growth, with new products and formulations constantly emerging and their applications in the biopharmaceutical field becoming increasingly widespread.

[0003] Low endotoxin levels are increasingly becoming a key quality attribute for products. However, the unique characteristics of biological products (different charges and spatial structures) and the complex composition of buffers (containing DTT, EDTA, glycerol, etc.) present new challenges for the accurate detection of endotoxins. For example, in endotoxin colorimetric assays, endotoxin recovery rates often fail to meet the acceptable standard of 50%-200%. Historical recovery data is shown in the table below:

[0004] Table 1: Recovery rate data of traditional methods

[0005] Experiment Date Sample Name Experimental methods Dilution factor Sample processing Spiked recovery rate R (%) 2024.09.02 CAS109 endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 38.2 2024.11.13 T7MP endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 54.4 2024.11.27 BSA endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 60.1 2024.12.23 NUC endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 54.4 2025.01.03 10×Transcription Buffer-A endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 4.55 2025.01.03 VCS endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 27.5 2025.04.09 MEH endpoint colorimetric method 100 Bacterial endotoxin test diluted with water 64.3

[0006] In this situation, traditional anti-interference strategies, such as dilution and high-temperature heating, cannot solve the problem. The changes in the product place new demands on current testing procedures and reagents. Summary of the Invention

[0007] The purpose of this invention is to provide a pretreatment kit and method for detecting endotoxins in biological products, so that the recovery rate of endotoxin detection samples of nuclease-based biological products can meet the standards for subsequent detection.

[0008] The present invention adopts the following technical solution:

[0009] This invention provides a pretreatment kit for detecting endotoxins in nuclease-based biological products, characterized by comprising:

[0010] Cracking agent A, neutralizing agent B, and stabilizer C;

[0011] Lysis agent A is a protein lysin.

[0012] Neutralizing agent B is a hydrogen peroxide solution.

[0013] Stability C is for magnesium sulfate solution.

[0014] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0015] The molar ratio of lysis agent A to nuclease in the sample to be tested is in the range of 1:25 to 1:100.

[0016] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0017] The operating temperature of pyrolysis agent A is 37℃-45℃.

[0018] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0019] The reaction time of pyrolysis agent A is ≥1h and ≤62h.

[0020] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0021] The pyrolysis termination condition is: 65℃ ≥30min

[0022] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0023] The molar ratio of neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10.

[0024] The conditions for neutralizing agent B to take effect are: shaking for 5-10 minutes.

[0025] Furthermore, the pretreatment kit for detecting endotoxins in nuclease-based biological products of the present invention also has the following characteristics:

[0026] The molar ratio of stabilizer C to EDTA is in the range of 1:1 to 1:10.

[0027] This invention also provides a pretreatment method for endotoxin detection samples of nuclease-based biological products, characterized by comprising the following steps:

[0028] Step 1: Take a predetermined amount of the sample to be tested, add an appropriate amount of pyrolysis agent A, incubate for a predetermined time, and then terminate the process;

[0029] Step 2: Add neutralizing agent B and shake to mix well;

[0030] Step 3: Dilute using stabilizer C.

[0031] Furthermore, the pretreatment method for endotoxin detection samples of nuclease-based biological products of the present invention also has the following characteristics:

[0032] The molar ratio of lysis agent A to nuclease protein in the sample is in the range of 1:25 to 1:100.

[0033] The operating temperature of pyrolysis agent A is 37℃-45℃;

[0034] The reaction time of pyrolysis agent A is: ≥1h, ≤62h.

[0035] The pyrolysis termination condition is: 65℃ ≥30min

[0036] Furthermore, the pretreatment method for endotoxin detection samples of nuclease-based biological products of the present invention also has the following characteristics:

[0037] The molar ratio of neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10.

[0038] Neutralizing agent B's action conditions: shake for 5-10 minutes.

[0039] The molar ratio of stabilizer C to EDTA is in the range of 1:1 to 1:10.

[0040] Beneficial effects of the invention: The pretreatment kit and pretreatment method provided by the present invention have been verified to be applicable to the pretreatment of various endotoxin detection methods such as gel electrophoresis, colorimetric assay, and recombinant factor C assay, thereby enabling the recovery rate of various nuclease biological products to meet the standards. Detailed Implementation

[0041] The technical solution of the present invention will be described in detail below through specific embodiments.

[0042] I. Main components of the pretreatment kit for endotoxin detection samples from nuclease-based biological products:

[0043] Lysis agent A: a protein lysin;

[0044] Neutralizing agent B: Hydrogen peroxide solution;

[0045] Stabilizer C: Magnesium sulfate solution.

[0046] name Manufacturer Item number Lysis agent A: proteinase K Thermo Fisher E00491 Neutralizing agent B: 30% hydrogen peroxide Sinopharm Chemical Reagent Co., Ltd. 10011218 Stabilizer C: Magnesium sulfate heptahydrate Sinopharm Chemical Reagent Co., Ltd. 10013018

[0047] Recovery rate refers to the ratio of the endotoxin content actually measured by detection methods to the known amount added, usually expressed as a percentage. Pharmacopoeia standards require a recovery rate between 50% and 200%.

[0048] The following embodiments determine the usage parameters of each component by controlling variables.

[0049] Example 1:

[0050] The molar ratio of lysis agent A to nuclease is 1:1.

[0051] The operating temperature of pyrolysis agent A is 37℃.

[0052] Duration of action: 1 hour

[0053] Termination condition: 65℃ for 30 min

[0054] Spike recovery rate R: 3.4%.

[0055] Example 2:

[0056] The molar ratio of lysis agent A to nuclease is 1:2.

[0057] The operating temperature of pyrolysis agent A is 37℃.

[0058] Duration of action: 1 hour

[0059] Termination condition: 65℃ for 30 min

[0060] Spiked recovery rate R: 21.9%.

[0061] Example 3:

[0062] The molar ratio of lysis agent A to nuclease is 1:25.

[0063] The operating temperature of pyrolysis agent A is 37℃.

[0064] Duration of action: 1 hour

[0065] Termination condition: 65℃ for 30 min

[0066] Spike recovery rate R: 104.8%.

[0067] Example 4:

[0068] The molar ratio of lysis agent A to nuclease is 1:50.

[0069] The operating temperature of pyrolysis agent A is 37℃.

[0070] Duration of action: 1 hour

[0071] Termination condition: 65℃ for 30 min

[0072] Spike recovery rate R: 89.5%.

[0073] Example 5:

[0074] The molar ratio of lysis agent A to nuclease is 1:100.

[0075] The operating temperature of pyrolysis agent A is 37℃.

[0076] Duration of action: 1 hour

[0077] Termination condition: 65℃ for 30 min

[0078] Spiked recovery rate R: 89.4%.

[0079] Based on the results of the above embodiments, Table 2 is prepared as follows:

[0080] Table 2: Effect of different ratios of lysing agent A and nuclease on spiked recovery rate

[0081] Molar ratio of lysis agent A to nuclease The operating temperature of pyrolysis agent A Duration of action Termination conditions Spiked recovery rate R (%) 1：1 37℃ 1h 65℃ for 30 minutes 3.4 1:2 37℃ 1h 65℃ for 30 minutes 21.9 1：25 37℃ 1h 65℃ for 30 minutes 104.8 1：50 37℃ 1h 65℃ for 30 minutes 89.5 1：100 37℃ 1h 65℃ for 30 minutes 89.4

[0082] Example 6:

[0083] The molar ratio of lysis agent A to nuclease is 1:25.

[0084] The operating temperature of pyrolysis agent A is 37℃.

[0085] Duration of action: 2 hours

[0086] Termination condition: 65℃ for 30 min

[0087] Spiked recovery rate R: 108.8%.

[0088] Example 7:

[0089] The molar ratio of lysis agent A to nuclease is 1:25.

[0090] The operating temperature of pyrolysis agent A is 37℃.

[0091] Duration of action: 4 hours

[0092] Termination condition: 65℃ for 30 min

[0093] Spiked recovery rate R: 108.8%.

[0094] Example 8:

[0095] The molar ratio of lysis agent A to nuclease is 1:25.

[0096] The operating temperature of pyrolysis agent A is 45℃.

[0097] Duration of action: 1 hour

[0098] Termination condition: 65℃ for 30 min

[0099] Spike recovery rate R: 99.0%.

[0100] Example 9:

[0101] The molar ratio of lysis agent A to nuclease is 1:25.

[0102] The operating temperature of pyrolysis agent A is 55℃.

[0103] Duration of action: 1 hour

[0104] Termination condition: 65℃ for 30 min

[0105] Spike recovery rate R: 71.0%.

[0106] Example 10:

[0107] The molar ratio of lysis agent A to nuclease is 1:25.

[0108] The operating temperature of pyrolysis agent A is 55℃.

[0109] Duration of action: 2 hours

[0110] Termination condition: 65℃ for 30 min

[0111] Spiked recovery rate R: 65.5%.

[0112] Example 11:

[0113] The molar ratio of lysis agent A to nuclease is 1:25.

[0114] The operating temperature of pyrolysis agent A is 37℃.

[0115] Duration of action: 62 hours

[0116] Termination condition: 65℃ for 30 min

[0117] Spike recovery rate R: 89.5%.

[0118] Based on embodiments 6 to 11, Table 3 is prepared as follows:

[0119] In Table 3, the data in the first row with an exposure time of 1 hour at 37°C are from Example 3.

[0120] Table 3: Effects of reaction temperature and time of pyrolysis agent A on spiked recovery rate

[0121] Molar ratio of lysis agent A to nuclease The operating temperature of pyrolysis agent A Duration of action Termination conditions Spiked recovery rate R (%) 1：25 37℃ 1h 65℃ for 30 minutes 104.8 1：25 37℃ 2h 65℃ for 30 minutes 108.8 1：25 37℃ 4h 65℃ for 30 minutes 99.6 1：25 45℃ 1h 65℃ for 30 minutes 99.0 1：25 55℃ 1h 65℃ for 30 minutes 71.0 1：25 55℃ 2h 65℃ for 30 minutes 65.5 1：25 37℃ 62h 65℃ for 30 minutes 89.5

[0122] Example 12:

[0123] The molar ratio of lysis agent A to nuclease is 1:25.

[0124] The operating temperature of pyrolysis agent A is 37℃.

[0125] Duration of action: 1 hour.

[0126] Termination condition: 65℃ for 30 minutes.

[0127] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:1.

[0128] Spike recovery rate R: 99.6%.

[0129] Example 13:

[0130] The molar ratio of lysis agent A to nuclease is 1:25.

[0131] The operating temperature of pyrolysis agent A is 37℃.

[0132] Duration of action: 1 hour.

[0133] Termination condition: 65℃ for 30 minutes.

[0134] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:10.

[0135] Spike recovery rate R: 101.1%.

[0136] Example 14:

[0137] The molar ratio of lysis agent A to nuclease is 1:25.

[0138] The operating temperature of pyrolysis agent A is 37℃.

[0139] Duration of action: 1 hour.

[0140] Termination condition: 65℃ for 30 minutes.

[0141] The molar ratio of neutralizing agent B to β-mercaptoethanol BME is 1:1.

[0142] Spiked recovery rate R: 98.6%.

[0143] Table 4: Effect of different ratios of neutralizing agent B and DTT on spiked recovery rate

[0144] Molar ratio of lysis agent A to nuclease The operating temperature of pyrolysis agent A Duration of action Termination conditions Dithiothreitol (DTT) or β-mercaptoethanol (BME) Spiked recovery rate R (%) 1：25 37℃ 1h 65℃ for 30 minutes Neutralizing agent B: DTT = 1:1 99.6 1：25 37℃ 1h 65℃ for 30 minutes Neutralizing agent B: DTT = 1:10 101.1 1：25 37℃ 1h 65℃ for 30 minutes Neutralizing agent B:BME = 1:1 98.6 1：25 37℃ 1h 65℃ for 30 minutes Neutralizing agent B: BME = 1:10 103.2

[0145] Example 15:

[0146] The molar ratio of lysis agent A to nuclease is 1:25.

[0147] The operating temperature of pyrolysis agent A is 37℃.

[0148] Duration of action: 1 hour.

[0149] Termination condition: 65℃ for 30 minutes.

[0150] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:1.

[0151] The molar ratio of stabilizer C to EDTA is 2:1.

[0152] Spike recovery rate R: 61.0%.

[0153] Example 16:

[0154] The molar ratio of lysis agent A to nuclease is 1:25.

[0155] The operating temperature of pyrolysis agent A is 37℃.

[0156] Duration of action: 1 hour.

[0157] Termination condition: 65℃ for 30 minutes.

[0158] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:1.

[0159] The molar ratio of stabilizer C to EDTA is 1:10.

[0160] Spike recovery rate R: 88.1%.

[0161] Example 17:

[0162] The molar ratio of lysis agent A to nuclease is 1:25.

[0163] The operating temperature of pyrolysis agent A is 37℃.

[0164] Duration of action: 1 hour.

[0165] Termination condition: 65℃ for 30 minutes.

[0166] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:1.

[0167] The molar ratio of stabilizer C to EDTA is 1:100.

[0168] Spike recovery rate R: 36.1%.

[0169] Table 5: Effect of different concentrations of magnesium ion solution on spike recovery rate

[0170] Molar ratio of lysis agent A to nuclease The operating temperature of pyrolysis agent A Duration of action Termination conditions The ratio of neutralizer B to DTT molar ratio of stabilizer C to EDTA Spiked recovery rate R (%) Example 15 1：25 37℃ 1h 65℃ for 30 minutes 1：1 2：1 61.0 Example 12 1：25 37℃ 1h 65℃ for 30 minutes 1：1 1：1 99.6 Example 16 1：25 37℃ 1h 65℃ for 30 minutes 1：1 1：10 88.1 Example 17 1：25 37℃ 1h 65℃ for 30 minutes 1：1 1：100 36.1

[0171] Table 5 uses data from Example 12, in which the molar ratio of stabilizer C to EDTA was 1:1.

[0172] As shown in Table 2, the molar ratio of lysis agent A to the nuclease of the target nuclease is between 1:25 and 1:100.

[0173] As shown in Table 3, the operating temperature of pyrolysis agent A is 37℃-45℃.

[0174] The reaction time of pyrolysis agent A is ≥1h and ≤62h.

[0175] Termination conditions: 65℃, ≥30min.

[0176] The molar ratio of neutralizing agent B to dithiothreitol (DTT) or β-mercaptoethanol (BME) is 1:1 to 1:10. This ratio is derived from the data in Table 4.

[0177] Neutralizer B usage conditions: Gently shake for 5-10 minutes.

[0178] See Table 5. The molar ratio of stabilizer C to EDTA ranges from 1:1 to 1:10.

[0179] Reagent kit usage instructions:

[0180] Step 1: Take a predetermined amount of the sample to be tested, which is 100 μL in this embodiment, add an appropriate amount of pyrolysis agent A, incubate, and then terminate.

[0181] Step 2: Add neutralizing agent B, shake to mix well.

[0182] Step 3: Dilute using stabilizer C.

[0183] The application effect of the pretreatment kit of the present invention in the endotoxin detection of common nucleic acid tool enzyme bioproducts:

[0184] The kit for pretreatment of endotoxin detection in common nucleic acid tool enzyme bioproducts of the present invention has been tested with extensive data and can be used with various endotoxin detection methods, including gel electrophoresis, colorimetric assay, and recombinant factor C assay. The experimental procedure follows the method described in Chinese Pharmacopoeia, Part IV, 1143. Experimental results are shown in Table 6.

[0185] Table 6: Comparative Experiment of the Application of the Kit in Different Endotoxin Detection Methods

[0186]

[0187] Extensive data testing has shown that this method can be applied to nuclease-based biological products such as CAS109, T7MP, BSA, NUC, 10×Transcription Buffer-A, VCS, and MEH. The experimental procedure follows the method described in Chinese Pharmacopoeia, Volume IV, Section 1143. The data are as follows:

[0188] Table 7: Comparative Experimental Results of the Kit's Application in Different Nuclease Products

[0189]

[0190] It is evident that the pretreatment kit and pretreatment method provided by this invention can be applied to the pretreatment of various endotoxin detection methods such as gel electrophoresis, colorimetric assay, and recombinant factor C assay, thereby enabling the recovery rate of various nuclease biological products to meet the standards.

Claims

1. A pretreatment kit for detecting endotoxins in nuclease-based biological products, characterized in that, include: Cracking agent A, neutralizing agent B, and stabilizer C; Lysis agent A is proteinase K, the molar ratio of lysis agent A to nuclease in the sample to be tested is 1:25, and the reaction time of lysis agent A is ≥1h and ≤2h. Neutralizing agent B is a hydrogen peroxide solution, and the molar ratio of neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:

10. The conditions for neutralizing agent B to take effect are: shaking for 5-10 minutes; The stability C is a magnesium sulfate solution, and the molar ratio of the stabilizer C to EDTA is in the range of 1:1 to 1:

10.

2. The pretreatment kit for detecting endotoxins in nuclease-based biological products as described in claim 1, characterized in that: The operating temperature of the pyrolysis agent A is 37℃-45℃.

3. The pretreatment kit for detecting endotoxins in nuclease-based biological products as described in claim 1, characterized in that: in, The pyrolysis termination conditions were: 65℃ for a duration of ≥30 min.

4. A pretreatment method for endotoxin detection samples of nuclease-based biological products, characterized in that, Includes the following steps: Step 1: Take a predetermined amount of the sample to be tested, add proteinase K at a molar ratio of 1:25 with the nuclease in the sample to be tested, incubate for ≥1h, ≤2h, then terminate the incubation. The termination conditions are: 65℃, duration ≥30min. Step 2: Add neutralizing agent B and shake to mix; the molar ratio of neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10; The conditions for the neutralizing agent B to take effect are: shaking for 5-10 minutes; Step 3: Dilute with stabilizer C, wherein the molar ratio of stabilizer C to EDTA is in the range of 1:1 to 1:10.

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