Method for detecting purity of enterokinase protein and application thereof

Abstract

This application relates to the field of drug detection technology, and provides a method for detecting the purity of enterokinase protein. The method uses octadecylsilane-bonded silica gel as the packing material, trifluoroacetic acid aqueous solution as mobile phase A, and trifluoroacetic acid acetonitrile solution as mobile phase B, employing a specific elution procedure. This method shortens the detection time while improving the separation effect of enterokinase protein components, resulting in good separation. The method is simple, and exhibits high precision, stability, and repeatability. Therefore, it can comprehensively and rapidly detect the purity of enterokinase protein, which is beneficial for the quality control of enterokinase and thus helps improve the safety and stability of the drug.

Description

Technical Field

[0001] This application relates to the field of drug testing, specifically to a method and application for detecting the purity of enterokinase protein. Background Technology

[0002] Enterokinase is a heterodimeric serine protease found in the duodenum of mammals. Currently, the purity of enterokinase protein is determined by SDS-PAGE non-reducing electrophoresis. This method can detect proteins of different molecular weights, and the purity of the protein is assessed by observing whether the protein bands after electrophoresis are clear and uniform. This method has high resolution and is simple to operate, but it is not sensitive to extremely small and large protein molecules, and protein aggregation may occur during electrophoresis, thus affecting the final result. Summary of the Invention

[0003] The problem this application aims to solve is that there is no publicly available method for effectively detecting the purity of enterokinase protein in the prior art; therefore, this application provides a method for detecting the purity of enterokinase protein.

[0004] Therefore, this application provides a method for detecting the purity of enterokinase protein, comprising the following steps:

[0005] After diluting the enterokinase sample to be tested into a sample solution, the sample solution was detected by high performance liquid chromatography.

[0006] The chromatographic conditions in high-performance liquid chromatography (HPLC) include using octadecylsilane-bonded silica gel as the stationary phase, trifluoroacetic acid aqueous solution as mobile phase A, and trifluoroacetic acid acetonitrile solution as mobile phase B, with gradient elution as specified in the table below:

[0007]

[0008] In some optional embodiments, the volume concentration of trifluoroacetic acid in the aqueous trifluoroacetic acid solution is 0.08-0.12%; the volume concentration of trifluoroacetic acid in the trifluoroacetic acid acetonitrile solution is 0.08-0.12%.

[0009] Perform gradient elution according to the specifications in the table below:

[0010]

[0011] In some optional implementations, the detection wavelength is 214-280 nm, and optionally, the detection wavelength is 214 nm.

[0012] In some optional embodiments, the chromatographic conditions are such that the injection volume of the sample solution is 5-100 μL, and optionally, the injection volume of the sample solution is 100 μL.

[0013] In some alternative implementations, the column temperature is 30-65°C, and optionally, the column temperature is 40°C;

[0014] And / or, the flow rate is 0.8-1.0 mL / min, optionally, the flow rate is 1.0 mL / min.

[0015] In some optional embodiments, the chromatographic conditions include a column with dimensions of 150 mm × 4.6 mm and a packing particle size of 5 μm.

[0016] In some optional embodiments, the sample solution is prepared by measuring the enterokinase sample to be tested and adding a solvent to prepare a sample solution;

[0017] Optionally, the mass ratio of the enterokinase sample to the volume of the solvent is 100-150:1; the mass-to-volume relationship is μg / mL; and / or, the solvent is any one of an aqueous glycerol solution, ultrapure water, an aqueous trifluoroacetic acid solution, an aqueous Tris solution, or a mixed solution of an aqueous Tris solution and glycerol, wherein the volume concentration of glycerol in the aqueous glycerol solution is 40-60%.

[0018] In some optional embodiments, the detection method further includes the step of using an aqueous glycerol solution as a reference solution and detecting the reference solution by high performance liquid chromatography in the detection method to obtain a reference chromatogram;

[0019] Optionally, the reference solution is an aqueous glycerol solution; the volume concentration of glycerol in the aqueous glycerol solution is 40-60%.

[0020] The technical solution of this application has the following advantages:

[0021] 1. The method for detecting the purity of enterokinase protein provided in this application uses octadecylsilane-bonded silica gel as the packing material, trifluoroacetic acid aqueous solution as mobile phase A, and trifluoroacetic acid acetonitrile solution as mobile phase B, and employs a specific elution procedure. This method not only shortens the detection time but also improves the separation effect of enterokinase protein components, resulting in good separation. The method is simple, and the detection method has high precision and good stability. Therefore, it can comprehensively and rapidly detect the purity of enterokinase protein, which is beneficial for the quality control of enterokinase and thus helps to improve the safety and stability of the drug. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a comparison of the characteristic chromatogram of the enterokinase sample in Example 1 of this application with the characteristic chromatogram of the control sample;

[0024] Figure 2 This is a chromatogram of the enterokinase sample from Example 1 of this application;

[0025] Figure 3 This is the chromatogram obtained after elution under the first gradient condition in Experimental Example 1 of this application;

[0026] Figure 4 This is the chromatogram obtained after elution under the second gradient condition in Experimental Example 1 of this application;

[0027] Figure 5 This is the chromatogram obtained after elution under the third gradient condition in Experiment Example 1 of this application;

[0028] Figure 6 Chromatogram of the blank reference solution under the third gradient condition in Experimental Example 1 of this application;

[0029] Figure 7 The chromatogram with a wavelength of 214 nm in Test Example 1 of this application;

[0030] Figure 8 This is the chromatogram of Test Example 1 of this application at a wavelength of 280 nm;

[0031] Figure 9 This is the chromatogram of Test Example 1 of this application with a column temperature of 40°C;

[0032] Figure 10 This is the chromatogram of the column temperature at 65°C in Test Example 1 of this application;

[0033] Figure 11 This is the chromatogram detected by column ① in Experimental Example 1 of this application;

[0034] Figure 12 This is the chromatogram detected by column ② in Test Example 1 of this application;

[0035] Figure 13 This is the chromatogram detected by column ③ in Test Example 1 of this application;

[0036] Figure 14 This is the chromatogram detected by column ④ in Test Example 1 of this application;

[0037] Figure 15 This is the chromatogram of the enterokinase sample diluted to 1.5 μg / ml in Test Example 1 of this application;

[0038] Figure 16 This is the chromatogram of the enterokinase sample diluted to 1.2 μg / ml in Test Example 1 of this application;

[0039] Figure 17 This is a chromatogram of the enterokinase sample diluted to 1.0 μg / ml in Test Example 1 of this application.

[0040] In the chromatogram above, the vertical axis represents absorbance (AU), and the horizontal axis represents time (minutes). Detailed Implementation

[0041] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone based on the teachings of this application or by combining features of this application with other prior art, falls within the scope of protection of this application. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. Percentages not specified in this application are volume percentages.

[0042] The instruments and reagents used in this application are as follows:

[0043] 1. Instruments and reagents

[0044] 1.1 Instruments

[0045] Chromatograph: Photodiode array detector (PDA detector);

[0046] Chromatographic column: ①Waters Symmetry300TM-C18, 4.6*150mm, 5μm;

[0047] ②Waters XBridge C18, 5μm, 4.6*250nm,

[0048] ③Agilent Zorbax SB-C3, 44.6*150mm, 5μm;

[0049] ④Zorbax Eclipse XDB-C18, 4.6*250mm, 5μm.

[0050] 1.2 Test Drugs

[0051] Glycerin (batch number: 20221207, purchased from Sinopharm Chemical Reagent Co., Ltd.)

[0052] Enterokinase sample (batch number: 20201219, purchased from Chengdu Yuangu Biotechnology Co., Ltd.);

[0053] 1.3 Reagents

[0054] Trifluoroacetic acid and acetonitrile were of chromatographic grade (Thermo Fisher, Merck), and water was ultrapure water; all other reagents were of analytical grade.

[0055] Example 1

[0056] A method for detecting the purity of enterokinase protein, comprising:

[0057] (1) Preparation of enterokinase sample solution: Accurately measure an appropriate amount of enterokinase sample, add 50% glycerol aqueous solution to make the enterokinase sample concentration 120-150 μg / ml, stir evenly, and the solution is obtained.

[0058] The enterokinase sample used in this embodiment was purchased from Chengdu Yuangu Biotechnology Co., Ltd., batch number: 20201219.

[0059] (2) Preparation of reference solution: Take a 50% glycerol aqueous solution as a blank reference solution.

[0060] (3) Detection by high performance liquid chromatography

[0061] Enterokinase solution and reference solution were detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: column: Symmetry 300. TM C18 5μm, 4.6×150mm; octadecylsilane-bonded silica gel as the packing material; gradient elution using 0.1% trifluoroacetic acid aqueous solution as mobile phase A and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B, as specified in Table 1; detection wavelength 214nm; flow rate 1.0ml / min; column temperature 40℃; injection volume 100μl; sample chamber temperature 10℃; recording time: 76min (with a 5min delay). The theoretical plate number, calculated based on the main peak of enterokinase protein, should be no less than 10,000.

[0062] Table 1 Gradient elution program

[0063]

[0064] The blank reference solution was analyzed using the above-described high-performance liquid chromatography (HPLC) method to obtain the characteristic chromatogram of the reference standard. The enterokinase sample was also analyzed using the same HPLC method to obtain the characteristic chromatogram of the enterokinase sample. The characteristic chromatograms of the enterokinase sample and the reference standard were compared. Figure 1 As shown, the retention time of enterokinase was 40.6 min. The enterokinase sample was analyzed using the above-described high-performance liquid chromatography (HPLC) method, and the characteristic chromatogram of the enterokinase sample is shown below. Figure 2As shown in the figure. The results showed that the purity of the chromatographic peaks of enterokinase was good. Among them, the chromatographic peak of enterokinase was peak 5, the retention time of enterokinase was 40.6 min, the resolution between enterokinase and adjacent peaks was 14.49, and the theoretical plate number was above 28800.

[0065] After HPLC analysis of the enterokinase sample, the chromatographic peak area of ​​enterokinase in the test solution was calculated, and the sum of the areas of each chromatographic peak in the test solution was calculated to obtain the total chromatographic peak area. Then, the chromatographic peak area of ​​enterokinase was divided by the total chromatographic peak area to obtain the percentage of the chromatographic peak area of ​​enterokinase to the total chromatographic peak area, which is the purity of enterokinase in the test sample. In this example, the purity of enterokinase in the enterokinase sample was 97.48%.

[0066] The above demonstrates that the detection method described in this embodiment can effectively obtain characteristic spectra with good separation of enterokinase. The detection method of this application can comprehensively and rapidly detect the purity of enterokinase protein, which is beneficial for the quality control of enterokinase and thus helps improve the safety and stability of the drug.

[0067] Experimental Example 1

[0068] 1. Establishment of chromatographic conditions

[0069] Preparation of sample solution: Accurately measure an appropriate amount of enterokinase sample, add 50% glycerol aqueous solution to make the concentration 120 μg / ml, stir well, and use as sample solution.

[0070] Preparation of reference solution: Take a 50% (v / v) aqueous solution of glycerol as the blank reference solution.

[0071] The sample solutions and control solutions in the following tests were prepared according to this procedure.

[0072] 1.1 Selection of elution gradient and flow rate

[0073] The sample solution was analyzed using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Symmetry 300 column. TM C18 column, 5 μm, 4.6 × 150 mm; octadecylsilane-bonded silica gel as packing material; mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution; gradient elution according to the gradient and flow rate specified in Table 3, 4, or 5; column temperature: 40 °C; injection volume: 100 μl; detection wavelength: 214 nm; sample chamber temperature: 10 °C.

[0074] Table 3 First-gradient elution conditions

[0075]

[0076] Table 4. Second gradient elution conditions

[0077] Time (min) Mobile phase A (Vol%) Mobile phase B (Vol%) Flow rate (ml / min) 0 100 0 0.8 5 75 25 0.8 20 40 60 0.8 25 20 80 0.8 30 100 0 0.8 35 100 0 0.8

[0078] Table 5 Elution conditions for the third gradient

[0079] Time (minutes) mobile phase Mobile phase B (Vol%) Flow rate (ml / min) 0～70 100 0→70 1.0 70～75 30→ 70→100 1.0 75～76 0→1 100→0 1.0

[0080] The chromatograms obtained after elution under the first gradient conditions in Table 3 are shown below. Figure 3 The chromatograms obtained after elution under the second gradient conditions in Table 4 are shown below. Figure 4 The chromatograms obtained after elution under the third gradient conditions in Table 5 are shown below. Figure 5 See Table 6.

[0081] Elution was performed using the first gradient elution condition in Table 3. The chromatogram showed significant baseline fluctuations, with the retention time of the main peak being close to that of the impurities introduced in the blank. Therefore, the gradient was softened, and elution was performed using the second gradient elution condition in Table 4. The results showed an uneven baseline. The inventors then lengthened the gradient and increased the flow rate to optimize the baseline fluctuations, and eluted according to the elution conditions in Table 5.

[0082] Meanwhile, the blank reference solution was detected using the above chromatographic conditions under the gradient conditions in Table 5, and the chromatogram is shown below. Figure 6 .

[0083] from Figure 3-6 The results showed that the various chromatograms were compared. Figure 5 The chromatographic peaks are rich in information, and the separation effect of the enterokinase protein chromatographic peak is good and the response is high. Therefore, this gradient condition was selected for subsequent research.

[0084] Table 6 System adaptability parameters for elution under three different gradient conditions

[0085]

[0086] 1.2 Selection of detection wavelength

[0087] The sample solution was analyzed using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Symmetry 300 column. TM C18 5μm, 4.6×150mm; octadecylsilane-bonded silica gel as the packing material; mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution; gradient elution according to the specifications in Table 1; flow rate: 1.0 ml / min; column temperature: 40℃; injection volume: 100 μl; sample chamber temperature: 10℃; recording time: 76 min (with a 5 min delay). Detection wavelength: 214 nm or 280 nm. Chromatogram at 214 nm is shown below. Figure 7 As shown, the chromatogram at a wavelength of 280 nm is as follows. Figure 8 As shown in Table 7, the comparison results between the two are presented in Table 7.

[0088] The detection results show that the peak height of the chromatographic peak at a wavelength of 214 nm is about 17 times higher than that at a wavelength of 280 nm. The sensitivity is higher at a wavelength of 214 nm, so 214 nm was chosen as the detection wavelength for further research.

[0089] Table 7 System adaptability parameters for different detection wavelengths

[0090]

[0091]

[0092] 1.3 Selection of different column temperatures

[0093] The effects of different column temperatures on the characteristic chromatograms of enterokinase were compared. Column temperatures were set at 40℃ or 65℃. High-performance liquid chromatography (HPLC) was used to detect the sample solutions under the following chromatographic conditions: Symmetry 300 column. TM A C18 column with a diameter of 5 μm and a diameter of 4.6 × 150 mm was used. Octadecylsilane-bonded silica gel was used as the packing material. Gradient elution was performed according to the specifications in Table 3, using a 0.1% (v / v) trifluoroacetic acid aqueous solution as mobile phase A and a 0.1% (v / v) trifluoroacetic acid acetonitrile solution as mobile phase B. The flow rate was 1.0 mL / min, the injection volume was 100 μL, the detection wavelength was 214 nm, and the sample chamber temperature was 10 °C. The effects of different column temperatures on the separation of enterokinase protein were observed using the above method.

[0094] The chromatogram at a column temperature of 40℃ is as follows Figure 9 The chromatogram for enterokinase retention time of 16.637 min and column temperature of 65℃ is as follows. Figure 10 The retention time of enterokinase was 16.251 min. The results indicate that changes in the relative positions of the impurity peak and the main peak can be observed under different temperature conditions. Increasing the column temperature reduced the main peak retention time by approximately 0.4 min, but this did not significantly alter the data or peak shape; therefore, a column temperature of 40℃ was chosen for further investigation.

[0095] 1.4 Selection of Different Chromatographic Columns

[0096] Enterokinase solution was analyzed using different brands of octadecylsilane-bonded silica columns (column ①: Waters Symmetry 300TM-C18, 4.6*150mm, 5μm; column ②: Waters XBridge C18, 5μm, 4.6*250nm; column ③: Agilent Zorbax SB-C3, 44.6*150mm, 5μm; column ④: Zorbax EclipseXDB-C18, 4.6*250mm, 5μm). The column used was Symmetry 300TM C18. 5 μm, 4.6 × 150 mm; octadecylsilane-bonded silica gel as the packing material; 0.1% trifluoroacetic acid aqueous solution as mobile phase A and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B, gradient elution according to the specifications in Table 5; flow rate 1.0 ml / min, injection volume 100 μl, detection wavelength 214 nm; column temperature 40℃; sample chamber temperature 10℃; recording time: 76 min (with a 5 min delay injection). Results are as follows: Figure 11-14 and Table 8; Figure 11 The chromatogram is for detection by column ①; Figure 12 The chromatogram is for detection by column ②; Figure 13 The chromatogram is for detection by column ③; Figure 14 This is the chromatogram detected by column ④.

[0097] The results showed that by comparing the characteristic chromatograms obtained from different brands of chromatographic columns, the baselines of columns ②, ③, and ④ all exhibited varying degrees of drift. The baseline of column ③ even showed a sharp drop at 23.4 min, while column ④ did not elute any peak except for enterokinase protein. Therefore, column ①, Symmetry C18, 4.6*150 mm, 5 μm column, is the preferred choice.

[0098] Table 8 System adaptability parameters for different chromatographic columns

[0099] Different chromatographic columns Retention time area high Resolution Tail factor Theoretical number of plates Chromatographic column ① 46.665 12487519 515515 28.58 1.07 28490.65 Column ② 31.198 2052118 61549 —— 0.99 20193.28 Column ③ 29.486 8516523 168793 —— 1.52 8561.363 Column ④ —— —— —— —— —— ——

[0100] 1.5 Determination of chromatographic conditions

[0101] Based on the above experiments, the chromatographic conditions for this application were determined as follows: Symmetry C18 column, 4.6*150mm, 5μm; octadecylsilane-bonded silica gel as the packing material; mobile phase A of 0.1% trifluoroacetic acid aqueous solution and mobile phase B of 0.1% trifluoroacetic acid acetonitrile solution, with gradient elution as specified in Table 5; flow rate of 1.0 ml / min; injection volume of 100 μl; detection wavelength of 214 nm; column temperature of 40℃; sample chamber temperature of 10℃; recording time of 76 min (including a 5 min delay injection).

[0102] Test Example 2: Determination of the detection limit

[0103] The enterokinase samples were detected according to the chromatographic conditions determined in Example 1.5. The enterokinase samples were diluted to 1.5 μg / ml, 1.2 μg / ml, and 1.0 μg / ml, respectively, and the results are as follows: Figure 15-17 As shown in Table 9.

[0104] Table 9. Detection of Enterokinase Samples at Different Concentrations

[0105]

[0106] The test results show that the signal-to-noise ratio is 5.52 at 1.5 μg / ml, 4.70 at 1.2 μg / ml, and not detected at 1.0 μg / ml. Therefore, the detection limit of the detection method of this application is 1.2 μg / ml.

[0107] Experimental Example 3

[0108] 1. Instrument precision

[0109] The enterokinase sample was tested under the chromatographic conditions determined in Example 1, Section 1.5. The enterokinase sample was diluted with the control solution to 150 μg / ml, and six parallel aliquots were prepared and tested separately to complete the repeatability experiment. On another day, another researcher used a different instrument to test the enterokinase sample under the chromatographic conditions determined in Example 1, Section 1.5. The enterokinase sample was diluted with the control solution to 120 μg / ml, and six parallel aliquots were prepared and tested separately to complete the intermediate precision determination. The experimental results are shown in Table 10 below:

[0110] Table 10 Precision Test Results

[0111]

[0112] The results show that the method has good precision.

[0113] 2. Durability

[0114] The enterokinase samples were detected under the chromatographic conditions determined in Example 1.5, which were considered standard conditions. Variations in column temperature (40℃ ± 2℃) and flow rate (1.0 ml / min ± 0.1 ml / min) were considered changes in conditions. The enterokinase samples were diluted with control solution to 120-150 μg / ml and detected under the specified chromatographic conditions. The experimental results are shown in Table 11 below.

[0115] Table 11 Durability Test Results

[0116]

[0117]

[0118] As can be seen from the table above, the testing method of this application has good robustness.

[0119] The above experiments show that the method for detecting the purity of enterokinase and its drug formulations provided in this application, using octadecylsilane-bonded silica gel as the packing material, trifluoroacetic acid aqueous solution as mobile phase A, and trifluoroacetic acid acetonitrile solution as mobile phase B, and employing a specific elution procedure, not only shortens the detection time but also improves the separation effect of enterokinase protein components. The method has good separation, is simple, and exhibits high precision, stability, and repeatability. Therefore, it can comprehensively and rapidly detect the purity and content of enterokinase protein, which is beneficial for the quality control of enterokinase and thus helps improve the safety and stability of the drug.

[0120] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. A method for detecting purity of an enterokinase protein, characterized by, Includes the following steps: The enterokinase sample to be tested was diluted into a sample solution and then detected by high performance liquid chromatography (HPLC). The detection wavelength was 214 nm. The batch number of the enterokinase sample was 20201219, and it was purchased from Chengdu Yuangu Biotechnology Co., Ltd. The chromatographic conditions in high-performance liquid chromatography (HPLC) include: a Waters Symmetry 300TM-C18 column; mobile phase A: trifluoroacetic acid aqueous solution; mobile phase B: trifluoroacetic acid acetonitrile solution; and mobile phase C: trifluoroacetic acid aqueous solution with a volume concentration of 0.08-0.12% and trifluoroacetic acid acetonitrile solution with a volume concentration of 0.08-0.12%. Perform gradient elution according to the specifications in the table below: 。 2. The method of claim 1, wherein the enterokinase protein purity is determined by, Under the specified chromatographic conditions, the injection volume of the sample solution is 5-100 µL.

3. The method of claim 2, wherein the enterokinase protein purity is determined by measuring the amount of enterokinase protein in the sample. Under the specified chromatographic conditions, the injection volume of the sample solution is 100 µL.

4. The method of claim 1, wherein the enterokinase protein purity is determined by measuring the amount of enterokinase protein in the sample. The column temperature is 30-65℃; and / or the flow rate is 0.8-1.0 mL / min.

5. The method for detecting the purity of enterokinase protein according to claim 1, characterized in that, The column temperature is 40°C; and / or the flow rate is 1.0 mL / min.

6. The method for detecting the purity of enterokinase protein according to claim 1, characterized in that, Under the chromatographic conditions, the column size was 150 mm × 4.6 mm, and the particle size of the packing material was 5 μm.

7. The method for detecting the purity of enterokinase protein according to claim 1, characterized in that, The sample solution is prepared by measuring the enterokinase sample to be tested and adding solvent to prepare the sample solution. The mass ratio of the enterokinase sample to the volume of the solvent is 100-150:1; the mass-to-volume relationship is μg / mL; and / or, the solvent is any one of glycerol aqueous solution A, ultrapure water, trifluoroacetic acid aqueous solution, Tris aqueous solution, or a mixed solution of Tris aqueous solution and glycerol, wherein the volume concentration of glycerol in glycerol aqueous solution A is 40-60%, and the final concentration of Tris in Tris aqueous solution is 20 mmol / L.

8. The method for detecting the purity of enterokinase protein according to any one of claims 1-7, characterized in that, The detection method further includes the step of using glycerol aqueous solution B as a reference solution and detecting the reference solution by high performance liquid chromatography according to any one of the detection methods of claims 1-7 to obtain a reference chromatogram; The volume concentration of glycerol in the glycerol aqueous solution B is 40-60%.

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