Pretreatment kit and pretreatment method of biological product endotoxin detection sample

Abstract

The invention provides a pretreatment kit and a pretreatment method for a biological product endotoxin detection sample, and the pretreatment kit for the nuclease biological product endotoxin detection sample is characterized by comprising a cracking agent A, a neutralizer B and a stabilizer C, the cracking agent A is protein lyase, the neutralizer B is a hydrogen peroxide solution, and the stabilizer C is a magnesium sulfate solution. The pretreatment kit and the pretreatment method provided by the invention can be applied to pretreatment of various endotoxin detection methods such as a gel method, a color development method and a recombinant C factor method, so that the recovery rate of various nuclease biological products reaches the standard.

Description

TECHNICAL FIELD

[0001] The present application relates to a pre-treatment kit for endotoxin detection samples of biological products, and also relates to a pre-treatment method for endotoxin detection samples of biological products, and belongs to the field of biological product detection. BACKGROUND

[0002] In the past decade, biological agent manufacturing has exploded, with new products, formulations constantly emerging and being used more and more widely in the field of biological pharmaceuticals.

[0003] Low endotoxin products have gradually become a key quality attribute of products, but due to the particularity of biological product products (different charges, spatial structures) and the complexity of Buffer components (containing DTT, EDTA, glycerol, etc.), new problems have been brought to the accurate detection of endotoxin. For example, in the endotoxin colorimetric method detection, a large number of endotoxin recovery rates do not meet the 50%-200% acceptable standard problem, the historical data of the recovery rate are as follows: Table 1: Recovery rate data of traditional method Date of experiment Sample name Method of experiment Dilution factor Sample treatment Recovery R(%) 2024.09.02 CAS109 Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 38.2 2024.11.13 T7MP Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 54.4 2024.11.27 BSA Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 60.1 2024.12.23 NUC Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 54.4 2025.01.03 10x Transcription Buffer-A Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 4.55 2025.01.03 VCS Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 27.5 2025.04.09 MEH Endpoint colorimetric method 100 Dilution with water for bacterial endotoxin test 64.3 In this case, it cannot be solved by traditional anti-interference strategies such as dilution, high-temperature heating, etc. The changes of the product put forward new requirements for the current detection process and reagent. SUMMARY

[0004] The purpose of the present application is to provide a pre-treatment kit and a pre-treatment method for endotoxin detection samples of biological products, so that the recovery rate of endotoxin detection samples of nuclease biological products reaches the standard of subsequent detection.

[0005] The present application adopts the following technical solutions: The present application provides a pre-treatment kit for endotoxin detection samples of nuclease biological products, characterized in that it comprises: a lysis agent A, a neutralizing agent B, and a stabilizer C; The lysis agent A is a protein lysing enzyme, The neutralizing agent B is a hydrogen peroxide solution, The stabilizer C is a magnesium sulfate solution.

[0006] Further, the pre-treatment kit for endotoxin detection samples of nuclease biological products of the present application also has the following characteristics: The molar ratio of the lysis agent A to the nuclease in the sample to be tested is in the range of 1:25 to 1:100.

[0007] Further, the pre-treatment kit for endotoxin detection samples of nuclease biological products of the present application also has the following characteristics: The action temperature of the lysing agent A is 37-45℃.

[0008] Further, the pretreatment kit for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The action time of the lysing agent A is ≥1h and ≤62h.

[0009] Further, the pretreatment kit for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The termination condition of the lysis is 65℃ for ≥30min. Further, the pretreatment kit for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The molar ratio of the neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10. The action condition of the neutralizing agent B is 5-10min of shaking.

[0010] Further, the pretreatment kit for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The molar ratio of the stabilizing agent C to EDTA is in the range of 1:1 to 1:10.

[0011] The present application further provides a pretreatment method for detecting endotoxin in a sample of a nuclease biological product, which is characterized by comprising the following steps: Step one: a predetermined amount of the sample to be detected is taken, and an appropriate amount of the lysing agent A is added, and after incubation for a predetermined time, the termination is performed. Step two: the neutralizing agent B is added, and shaking is performed for mixing. Step three: the stabilizing agent C is used for dilution.

[0012] Further, the pretreatment method for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The molar ratio of the lysing agent A to the nuclease protein in the sample is in the range of 1:25 to 1:100. The action temperature of the lysing agent A is 37-45℃. The action time of the lysing agent A is ≥1h and ≤62h.

[0013] The termination condition of the lysis is 65℃ for ≥30min. Further, the pretreatment method for detecting endotoxin in a sample of a nuclease biological product of the present application further has the following characteristics: The molar ratio of the neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10.

[0014] The working condition of neutralizing agent B: shaking for 5-10 min.

[0015] The molar ratio of stabilizer C to EDTA is in the range of 1:1 to 1:10.

[0016] Advantages of the application: The pre-treatment kit and pre-treatment method provided by the application can be applied to the pre-treatment of various endotoxin detection methods such as gel method, color development method, and recombinant C factor method, so that the recovery rate of various nuclease biological products meets the standard. DETAILED DESCRIPTION

[0017] The technical solutions of the application are described in detail below through specific embodiments.

[0018] I. Main components of the pre-treatment kit for endotoxin detection samples of nuclease biological products: Lysis agent A: a protein lysis enzyme; Neutralizing agent B: hydrogen peroxide solution; Stabilizer C: magnesium sulfate solution.

[0019] Name Manufacturer Catalogue number Lysis agent A: Proteinase K Thermo E00491 Neutralizing agent B: 30% hydrogen peroxide National Pharmaceutical Group Chemical Reagent Co., Ltd. 10011218 Stabilizing agent C: Magnesium sulfate heptahydrate, National Pharmaceutical Group Chemical Reagent Co., Ltd. 10013018 Recovery rate refers to the ratio of the actual measured endotoxin content to the known added amount, usually expressed as a percentage. The pharmacopoeia standard requires that the recovery rate should be between 50% and 200%.

[0020] The following examples determine the use parameters of each component by controlling variables.

[0021] Example 1:

[0022] The molar ratio of lysis agent A to nuclease is 1:1.

[0023] The action temperature of lysis agent A is 37°C.

[0024] Action time: 1h Termination condition: 65°C for 30 min Spiked recovery rate R: 3.4%.

[0025] Example 2:

[0026] The molar ratio of lysis agent A to nuclease is 1:2.

[0027] The action temperature of lysis agent A is 37°C.

[0028] Action time: 1h Termination condition: 65°C for 30 min Spiked recovery rate R: 21.9%.

[0029] Example 3:

[0030] Molar ratio of cleaving agent A to nuclease: 1 :25.

[0031] Temperature of cleaving agent A: 37°C.

[0032] Time of action: 1 h Termination condition: 65°C for 30 min Recovery rate R: 104.8%.

[0033] Example 4:

[0034] Molar ratio of cleaving agent A to nuclease: 1 :50.

[0035] Temperature of cleaving agent A: 37°C.

[0036] Time of action: 1 h Termination condition: 65°C for 30 min Recovery rate R: 89.5%.

[0037] Example 5:

[0038] Molar ratio of cleaving agent A to nuclease: 1 :100.

[0039] Temperature of cleaving agent A: 37°C.

[0040] Time of action: 1 h Termination condition: 65°C for 30 min Recovery rate R: 89.4%.

[0041] Based on the results of the above examples, Table 2 was prepared as follows: Table 2: Effect of different ratios of cleaving agent A to nuclease on recovery rate Molar ratio of lysis agent A to nuclease Temperature of lysis agent A Action time Termination condition Recovery R(%) 1：1 37℃ 1h 65℃ 30min 3.4 1:2 37℃ 1h 65℃ 30min 21.9 1：25 37℃ 1h 65℃ 30min 104.8 1：50 37℃ 1h 65℃ 30min 89.5 1：100 37℃ 1h 65℃ 30min 89.4 Example 6:

[0042] Molar ratio of cleaving agent A to nuclease: 1 :25.

[0043] Temperature of cleaving agent A: 37°C.

[0044] Time of action: 2 h Termination condition: 65°C for 30 min Recovery rate R: 108.8%.

[0045] Example 7:

[0046] Molar ratio of cleaving agent A to nuclease: 1 :25.

[0047] Temperature of cleaving agent A: 37°C.

[0048] Time of action: 4 h Termination condition: 65 °C 30 min Spiked recovery R: 108.8%.

[0049] Example 8:

[0050] Molar ratio of cleaving agent A to nuclease: 1 :25.

[0051] Cleaving agent A action temperature: 45 °C.

[0052] Action time: 1 h Termination condition: 65 °C 30 min Spiked recovery R: 99.0%.

[0053] Example 9:

[0054] Molar ratio of cleaving agent A to nuclease: 1 :25.

[0055] Cleaving agent A action temperature: 55 °C.

[0056] Action time: 1 h Termination condition: 65 °C 30 min Spiked recovery R: 71.0%.

[0057] Example 10: Molar ratio of cleaving agent A to nuclease: 1 :25.

[0058] Cleaving agent A action temperature: 55 °C.

[0059] Action time: 2 h Termination condition: 65 °C 30 min Spiked recovery R: 65.5%.

[0060] Example 11: Molar ratio of cleaving agent A to nuclease: 1 :25.

[0061] Cleaving agent A action temperature: 37 °C.

[0062] Action time: 62 h Termination condition: 65 °C 30 min Spiked recovery R: 89.5%.

[0063] In summary, Examples 6 to 11, Table 3 is made as follows: Wherein, the data of the first row in Table 3, 37 °C action time 1 h, uses the data of Example 3.

[0064] Table 3: Effect of cleaving agent A action temperature and time on spiked recovery Molar ratio of lysis agent A to nuclease Temperature of lysis agent A Action time Termination condition Recovery R(%) 1：25 37℃ 1h 65℃ 30min 104.8 1：25 37℃ 2h 65℃ 30min 108.8 1：25 37℃ 4h 65℃ 30min 99.6 1：25 45℃ 1h 65℃ 30min 99.0 1：25 55℃ 1h 65℃ 30min 71.0 1：25 55℃ 2h 65℃ 30min 65.5 1：25 37℃ 62h 65℃ 30min 89.5 Example 12: The molar ratio of lysis agent A to nuclease is 1:25.

[0065] The operating temperature of pyrolysis agent A is 37℃.

[0066] Duration of action: 1 hour.

[0067] Termination condition: 65℃ for 30 minutes.

[0068] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:1.

[0069] Spike recovery rate R: 99.6%.

[0070] Example 13: The molar ratio of lysis agent A to nuclease is 1:25.

[0071] The operating temperature of pyrolysis agent A is 37℃.

[0072] Duration of action: 1 hour.

[0073] Termination condition: 65℃ for 30 minutes.

[0074] The molar ratio of neutralizing agent B to dithiothreitol (DTT) is 1:10.

[0075] Spike recovery rate R: 101.1%.

[0076] Example 14: The molar ratio of lysis agent A to nuclease is 1:25.

[0077] The operating temperature of pyrolysis agent A is 37℃.

[0078] Duration of action: 1 hour.

[0079] Termination condition: 65℃ for 30 minutes.

[0080] The molar ratio of neutralizing agent B to β-mercaptoethanol BME is 1:1.

[0081] Spike recovery rate R: 98.6%.

[0082] Table 4: Effect of different ratios of neutralizing agent B and DTT on spiked recovery rate Molar ratio of lysis agent A to nuclease Temperature of lysis agent A Action time Termination condition Dithiothreitol (DTT) or β-mercaptoethanol (BME) Recovery R(%) 1：25 37℃ 1h 65℃ 30min Neutralizing agent B: DTT = 1:1 99.6 1：25 37℃ 1h 65℃ 30min Neutralizing agent B: DTT = 1:10 101.1 1：25 37℃ 1h 65℃ 30min Neutralizing agent B: BME = 1:1 98.6 1：25 37℃ 1h 65℃ 30min Neutralizing agent B: BME = 1:10 103.2 Example 15: The molar ratio of lysis agent A to nuclease is 1:25.

[0083] The operating temperature of pyrolysis agent A is 37℃.

[0084] Duration of action: 1 hour.

[0085] Termination condition: 65°C for 30 min.

[0086] Molar ratio of neutralizing agent B to dithiothreitol (DTT): 1:1.

[0087] Molar ratio of stabilizer C to EDTA: 2:1. Spiked recovery rate R: 61.0%.

[0088] Example 16: Molar ratio of lysing agent A to nuclease: 1:25.

[0089] Temperature for lysing agent A: 37°C.

[0090] Reaction time: 1 h.

[0091] Termination condition: 65°C for 30 min.

[0092] Molar ratio of neutralizing agent B to dithiothreitol (DTT): 1:1.

[0093] Molar ratio of stabilizer C to EDTA: 1:10.

[0094] Spiked recovery rate R: 88.1%.

[0095] Example 17: Molar ratio of lysing agent A to nuclease: 1:25.

[0096] Temperature for lysing agent A: 37°C.

[0097] Reaction time: 1 h.

[0098] Termination condition: 65°C for 30 min.

[0099] Molar ratio of neutralizing agent B to dithiothreitol (DTT): 1:1.

[0100] Molar ratio of stabilizer C to EDTA: 1:100.

[0101] Spiked recovery rate R: 36.1%.

[0102] Table 5: Effect of different concentrations of magnesium ion solution on spiked recovery rate

[0103] Molar ratio of lysis agent A to nuclease Temperature of lysis agent A Action time Termination condition Ratio of neutralizing agent B and DTT Molar ratio of stabilizing agent C and EDTA Recovery R(%) Example 15 1：25 37℃ 1h 65℃ 30min 1：1 2：1 61.0 Example 12 1：25 37℃ 1h 65℃ 30min 1：1 1：1 99.6 Example 16 1：25 37℃ 1h 65℃ 30min 1：1 1：10 88.1 Example 17 1：25 37℃ 1h 65℃ 30min 1：1 1：100 36.1 The data of Example 12, in which the molar ratio of stabilizer C to EDTA was 1:1, were used in Table 5.

[0104] As shown in Table 2, the molar ratio of lysing agent A to the nuclease to be detected was between 1:25 and 1:100.

[0105] The action temperature of the lysing agent A is 37-45 DEG C, as shown in Table 3. The action time of the lysing agent A is greater than or equal to 1 hour and less than or equal to 62 hours.

[0106] The termination condition is 65 DEG C for greater than or equal to 30 minutes.

[0107] The molar ratio of the neutralizing agent B to dithiothreitol (DTT) or beta-mercaptoethanol (BME) is 1:1 to 1:10. The ratio is obtained according to the data in Table 4.

[0108] The use condition of the neutralizing agent B is slight shaking for 5-10 minutes.

[0109] Referring to Table 5, the molar ratio of the stabilizing agent C to EDTA ranges from 1:1 to 1:10.

[0110] The kit use process is as follows: Step one: a predetermined amount of a sample to be tested, 100 ul in the embodiment, is added with an appropriate amount of the lysing agent A, and after incubation, termination is performed.

[0111] Step two: the neutralizing agent B is added, and shaking is performed for mixing, Step three: the stabilizing agent C is used for dilution.

[0112] The application effect of the pretreatment kit of the application in the endotoxin detection of common nucleic acid tool enzyme biological products is as follows: The pretreatment kit of the application for the endotoxin detection of common nucleic acid tool enzyme biological products can be applied to various endotoxin detection methods such as gel method, colorimetric method and recombinant C factor method, through a large amount of data testing. The experimental process adopts the method recorded in Chinese Pharmacopoeia Part IV 1143. The experimental results are shown in Table 6.

[0113] Table 6: Comparison experiment of the application of the kit in different endotoxin detection methods

[0114] Through a large amount of data testing, the kit can be applied to CAS109, T7MP, BSA, NUC, 10x Transcription Buffer-A, VCS, MEH and other nuclease biological products. The experimental process adopts the method recorded in Chinese Pharmacopoeia Part IV 1143. The data are as follows: Table 7: Comparison experiment results of the application of the kit in different nuclease products

[0115] It can be seen that the pretreatment kit and the pretreatment method provided by the application can be applied to the pretreatment of various endotoxin detection methods such as gel method, colorimetric method and recombinant C factor method, so that the recovery rate of various nuclease biological products meets the standard.

Claims

1. A pre-treatment kit for detecting endotoxin in a sample of a nuclease class biological product, characterized in that, Comprising: lytic agent A, neutralizing agent B and stabilizing agent C; lytic agent A is proteinase k, the molar ratio of which to the nuclease in the sample to be tested is in the range of 1:25 to 1:100, the action time of the lytic agent A is ≥1h, ≤62h; neutralizing agent B is hydrogen peroxide solution, stabilizing agent C is magnesium sulfate solution.

2. The pretreatment kit for detecting samples of endotoxins in nucleases biological products according to claim 1, characterized in that: the action temperature of the lytic agent A is 37-45℃.

3. The pretreatment kit for detecting samples of endotoxins in nucleases biological products according to claim 1, characterized in that: wherein the termination condition of lysis is 65℃ ≥30min.

4. The pretreatment kit for detecting samples of endotoxins in nucleases biological products according to claim 1, characterized in that: wherein, the molar ratio of the neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10, the action condition of the neutralizing agent B is 5-10min of shaking.

5. The pretreatment kit for detecting samples of endotoxins in nucleases biological products according to claim 1, characterized in that: wherein the molar ratio of the stabilizing agent C to EDTA is in the range of 1:1 to 1:

10.

6. A method for the pre-treatment of a sample for endotoxin detection in a nuclease class biological product, characterized by, Comprising the following steps: Step one: take a predetermined amount of sample to be tested, add proteinase k with a molar ratio to the nuclease in the sample to be tested in the range of 1:25 to 1:100, incubate for ≥1h, ≤62h, then terminate, the termination condition is 65℃ ≥30min; Step two: add neutralizing agent B and shake to mix; Step three: dilute with stabilizing agent C.

7. The pretreatment method for detecting samples of endotoxins in nucleases biological products according to claim 6, characterized in that: wherein the molar ratio of the neutralizing agent B to dithiothreitol or β-mercaptoethanol is in the range of 1:1 to 1:10; the action condition of the neutralizing agent B is 5-10min of shaking; the molar ratio of the stabilizing agent C to EDTA is in the range of 1:1 to 1:10.

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