Preparation method and detection method of detection sample for host cell protein residues

Abstract

The invention relates to biological product detection, in particular to a preparation and detection method of a host cell protein residue detection sample. After a test sample is treated by trypsin for a short time, ultrafiltration separation is carried out by utilizing the difference of molecular weight, a peptide fragment zymolyte is obtained by carrying out enzymolysis on host cell protein, and the peptide fragment zymolyte is fully subjected to overnight enzymolysis by trypsin, so that a detection sample without antibody influence can be obtained. A standard curve is quantitatively drawn through an external standard method, peptide fragments and concentrations in a detection sample are detected through liquid chromatography-mass spectrometry, the types of host cell proteins and the corresponding concentrations of the proteins are obtained by combining the standard curve, and the method has a large dynamic detection range.

Description

Technical Field

[0001] The present invention relates to biological product detection, and in particular to a method for preparing a detection sample of host cell protein residues and a method for detecting the same. Background Art

[0002] Host cell protein (HCP) is a process-related impurity generated during the production of recombinant biotherapeutics. Despite multiple downstream purification stages, it remains at trace levels. The concentration of residual HCP in the preparation is often in the range of 1ppm to 100ppm, or even below the sub-ppm level, which may affect the quality, stability, efficacy or safety of the product. Enzyme-linked immunosorbent assay (ELISA) kits are often used to detect HCP residues. ELISA has good sensitivity, selectivity, dynamic range, sample throughput capacity and cost efficiency. However, ELISA relies on immune response and is only used to detect specific or total HCP levels. It cannot detect non-immunoreactive proteins or identify individual proteins.

[0003] In order to obtain the types of host cell proteins remaining in the sample and the content of various proteins, conventional methods for detecting protein types and contents can be used, such as chromatography. However, this method of widely detecting multiple types of proteins usually requires that the content of each protein is similar. The antibody content in the sample for host cell protein residue detection is much higher than the host cell protein content, which will have a significant adverse effect on the detection of host cell proteins. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a test sample and a method for detecting host cell protein residues that can eliminate the influence of antibodies.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a test sample for host cell protein residues, comprising:

[0007] Performing an enzymatic hydrolysis treatment on a product sample containing host cell proteins and antibodies by trypsin for a first period of time to obtain a preliminary enzymatic hydrolyzate, wherein the preliminary enzymatic hydrolyzate includes a first peptide group obtained by enzymatic hydrolysis of the host cell protein and the antibody;

[0008] Ultrafiltration treatment of the preliminary enzymatic hydrolysate to obtain a filtrate, wherein the cutoff of the ultrafiltration treatment is a first cutoff, the first cutoff is greater than the molecular weight of the peptides in the first peptide group, and the first cutoff is less than the molecular weight of the antibody;

[0009] The filtrate is enzymatically treated with trypsin, and the reaction is terminated after a second period of time to obtain the test sample.

[0010] Optionally, the first time is any value between 2.5 h and 3.5 h, and the first retention amount is any value between 80 kD and 130 kD.

[0011] Optionally, the ultrafilter is an ultrafiltration tube, and filtration with the ultrafiltration tube includes centrifugation at a speed of any value between 10000g and 15000g for a time of any value between 20min and 40min.

[0012] Optionally, the mass ratio of the trypsin used to process the product sample to the product sample is 1: any value in the range of (30-100), and the mass ratio of the trypsin used to process the filtrate to the product sample is 1: any value in the range of (30-100).

[0013] Optionally, after the filtrate is enzymatically hydrolyzed by the trypsin and the reaction is terminated, a desalting treatment is performed.

[0014] In a second aspect, the present invention further provides a method for detecting host cell protein residues, comprising:

[0015] Detecting the protein types and corresponding detection response values ​​in the test sample prepared by the above-mentioned preparation method by liquid chromatography-mass spectrometry;

[0016] A plurality of external standard samples are prepared by the same method as the preparation method of the test sample to obtain a plurality of external standard test samples, and the external standard response values ​​of the plurality of external standard test samples are detected by the liquid chromatography-mass spectrometry, and an external standard curve is drawn, wherein the plurality of external standard test samples respectively include labeled peptide segments with different concentrations, and the labeled peptide segments are obtained by hydrolyzing labeled proteins with different concentrations, the molecular weight of the labeled peptide segment is less than the first retention amount, and the host cell protein does not contain the labeled protein;

[0017] The detection concentration is obtained according to the detection response value and the external standard curve.

[0018] Optionally, the marker protein includes one or more of horse cytochrome C, horse myoglobin, bovine β-lactoglobulin, bovine serum albumin, β-casein, and chicken lysozyme.

[0019] Optionally, a labeled protein is quantitatively added to the preliminary enzymatic hydrolysis solution, and the external standard sample is obtained by mixing a plurality of the product samples.

[0020] Optionally, the external standard samples are the marker proteins of different masses.

[0021] Optionally, the test sample is freeze-dried and then re-dissolved in an organic solvent of a mobile phase in the liquid chromatography-mass spectrometry detection, and then the liquid chromatography-mass spectrometry detection is performed.

[0022] According to the first aspect of the present invention, the product sample is treated with trypsin to rapidly hydrolyze the host cell protein. Since the antibody has a strong resistance to trypsin, when the host cell protein undergoes a certain degree of hydrolysis, the antibody still does not undergo hydrolysis, or only undergoes a small amount of hydrolysis. At this time, the antibody is filtered through an ultrafilter with a first interception amount, intercepting the antibody, and filtering the first peptide segment group formed by the host cell protein, so that the two amounts of antibody-related peptide segments in the test sample are greatly reduced to a level similar to the amount of host cell protein, thereby eliminating the effect of the antibody on the HCP detection of the antibody in the product sample. After the filtration is completed, the filtrate is hydrolyzed again, and the generation of false positive results during the test is reduced by sufficient hydrolysis. Performing the hydrolysis twice can improve the degree of separation of the antibody from the host cell protein and its related peptide segments, reduce the residue of the antibody-related peptide segments after the ultrafiltration is completed, and reduce the loss of the first peptide segment group, which helps to improve the accuracy of the test results.

[0023] According to a second aspect of the present invention, the type and content of peptide segments in a test sample containing a quantitative marker protein are detected by liquid chromatography-mass spectrometry (LC-MS), and the type and content of the protein are analyzed, which has low cost, simple operation, can obtain more protein information, and has high accuracy. Drawing a standard curve by an external standard method and calculating the content of each protein in combination with the standard curve helps to improve the accuracy of the detection.

[0024] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the method for preparing a sample for detecting host cell protein residues as shown in Example 1 of the present invention;

[0026] Figure 2 This is a flow chart of the method for detecting host cell protein residues shown in Example 1 of the present invention;

[0027] Figure 3 This is the working curve shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0030] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0031] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] See also Figure 1 The method for preparing a sample for detecting host cell protein residues to be protected by the present invention comprises:

[0033] S1. Performing enzymatic hydrolysis treatment on a product sample containing host cell proteins and antibodies by trypsin for a first period of time to obtain a preliminary enzymatic hydrolyzate, which includes a first peptide group and antibodies obtained by proteolysis of the host cell protein.

[0034] S2. Ultrafiltration is performed on the preliminary enzymatic hydrolyzate to obtain a filtrate. The retention amount of the ultrafiltration treatment is a first retention amount. The first retention amount is greater than the molecular weight of the peptides in the first peptide group, and the first retention amount is less than the molecular weight of the antibody.

[0035] S3. Performing enzymatic hydrolysis on the filtrate by using trypsin, and terminating the reaction after a second period of time to obtain a test sample.

[0036] The product sample is treated with trypsin to rapidly hydrolyze the host cell protein. Since the antibody has a strong resistance to the host cell protein, when the host cell protein undergoes a certain degree of hydrolysis, the antibody still does not undergo hydrolysis, or only undergoes a small amount of hydrolysis. At this time, the antibody is filtered through an ultrafilter with a first interception amount, intercepting the antibody and filtering the first peptide group formed by the host cell protein, so that the amount of antibody-related peptides in the test sample is greatly reduced to a level similar to the amount of host cell protein, thereby eliminating the effect of the antibody on the HCP detection in the product sample. After the filtration is completed, the filtrate is hydrolyzed again, and the generation of false positive results during the test is reduced by sufficient hydrolysis. Performing hydrolysis twice can improve the degree of separation of antibodies from host cell proteins and their related peptides, reduce the residue of antibody-related peptides after ultrafiltration, and reduce the loss of the first peptide group, which helps to improve the accuracy of the test results.

[0037] In some embodiments, the first time in step S1 is any value in 2.5h to 3.5h, for example, it can be any value in 2.5h, 2.7h, 2.9h, 3.1h, 3.3h and 3.5h. The first retention amount in step S2 is any value in 50kD to 100kD, for example, it can be any value in 50kD, 60kD, 70kD, 80kD, 90kD and 100kD. The resistance of most antibodies to trypsin and the enzymatic hydrolysis rate when trypsin treats other non-antibody proteins are detected. It is finally found that after the above-mentioned first time of trypsin treatment of the antibody, the antibody hardly undergoes enzymatic hydrolysis, while other non-antibody proteins are initially enzymatically hydrolyzed, and the molecular weight of most peptide chain fragments is less than 50kD. Since the molecular weight of the antibody is about 150kD, selecting an ultrafilter with a first retention amount helps to fully filter out the antibody, and reduces the loss of the first peptide segment group, which helps to improve the accuracy of subsequent detection.

[0038] In some embodiments, the ultrafilter is an ultrafiltration tube, and filtering with the ultrafiltration tube includes centrifuging at a speed of any value between 10000g and 15000g for a time of any value between 20min and 40min. The speed can be, for example, any value between 10000g, 11000g, 12000g, 13000g, 14000g and 15000g, and the centrifugation time can be, for example, any value between 20min, 25min, 30min, 35min and 40min.

[0039] In some embodiments, the mass ratio of trypsin used to treat the product sample in step S1 to the product sample is any value in the range of 1:(30-100), for example, it can be any value in the range of 1:30, 1:40, 1:60, 1:80 and 1:100. The mass ratio of trypsin used to treat the filtrate in step S3 to the product sample is any value in the range of 1:(30-100), for example, it can be any value in the range of 1:30, 1:40, 1:60, 1:80 and 1:100. Limiting the mass ratio of trypsin to the sample helps reduce the risk of over-enzyme hydrolysis or insufficient enzymolysis.

[0040] In some embodiments, after the filtrate is enzymatically treated with trypsin and the reaction is terminated in step S3, a desalting treatment is performed to help prevent ions in the filtrate from adversely affecting subsequent detection, such as forming impurity peaks.

[0041] The method for detecting host cell protein residues claimed in the present invention comprises:

[0042] S4. Detect the protein types and corresponding detection response values ​​in the test sample prepared by the methods in S1 to S3 by liquid chromatography-mass spectrometry.

[0043] S5. Prepare multiple external standard samples by the same method as in S4 to obtain multiple external standard detection samples, and detect the external standard response values ​​of the multiple external standard detection samples by liquid chromatography-mass spectrometry, and draw external standard curves. The multiple external standard detection samples respectively include labeled peptides with different concentrations, and the labeled peptides are obtained by hydrolyzing labeled proteins with different concentrations. The molecular weight of the labeled peptides is less than the first retention amount, and the host cell proteins do not contain labeled proteins.

[0044] S6. Determine the detection concentration based on the detection response value and the external standard curve.

[0045] Liquid chromatography-mass spectrometry (LC-MS) is used to detect the type and content of peptides in the test sample containing quantitative marker proteins, and the type and content of proteins are analyzed. It has low cost, simple operation, and high accuracy. The quantification of proteins by external standard method is relatively convenient as a whole. Drawing a standard curve and calculating the content of each protein in combination with the standard curve can help improve the accuracy of the test. Selecting a marker protein with a molecular weight less than the first retention amount can prevent a large amount of marker protein from being lost during the filtration process.

[0046] In some embodiments, the marker protein includes one or more of horse cytochrome C, horse myoglobin, bovine β-lactoglobulin, bovine serum albumin, β-casein, and chicken lysozyme. The molecular weight of these proteins is about 75kD, and they are not contained in common host cells, and have high universality.

[0047] In some embodiments, a labeled protein is quantitatively added to the preliminary enzymatic hydrolysis solution, and the external standard sample is obtained by mixing several product samples.

[0048] By adding the labeled protein as an internal standard to the product sample and making the components of the external standard sample similar to those of the product sample, the difference in matrix effect between the product sample and the external standard sample in LC-MS detection is reduced, thereby improving the accuracy of the detection results. Adding the labeled protein to the product sample can easily lead to protein-protein interaction between the labeled protein and the product sample, affecting the external standard effect and being detrimental to the qualitative and quantitative analysis of the product sample itself. Adding the labeled protein to the preliminary enzymatic hydrolysis solution allows the labeled protein to be pre-treated together with the product sample to control variables and avoid protein-protein interaction.

[0049] In some embodiments, the external standard samples are labeled proteins of different masses. The labeled proteins are not added when preparing the test samples. The samples are only measured by the external standard method, and the standard curve is repeatedly used. The steps are simple and the cost is low.

[0050] In some embodiments, the test sample is freeze-dried and then re-dissolved in an organic solvent of a mobile phase in a liquid chromatography-mass spectrometry test before being subjected to liquid chromatography-mass spectrometry detection, which helps reduce interference from the solvent in the test sample.

[0051] Please refer to the following examples for details.

[0052] Embodiment 1:

[0053] After the antibody product is prepared, it is necessary to carry out purification steps such as affinity chromatography, low pH virus inactivation, intermediate sample deep filtration, anion chromatography, cation chromatography and ultrafiltration in sequence. In this embodiment, the intermediate products obtained in each step are taken as product samples, which are marked as AC group, VIN group, IDF group, AEX group, CEX group and UF group in sequence, and a sample is taken from each group to prepare a test sample for host cell protein residues, and the detection of host cell protein residues in the product samples is realized by the test samples.

[0054] See also Figure 1 A method for preparing a host cell protein residue detection sample shown in a preferred embodiment of the present application includes:

[0055] S1. Performing enzymatic hydrolysis treatment on a product sample containing host cell proteins and antibodies by trypsin for a first period of time to obtain a preliminary enzymatic hydrolyzate, which includes a first peptide group and antibodies obtained by proteolysis of the host cell protein.

[0056] S2. Filtering the preliminary enzymatic hydrolyzate through an ultrafilter having a first cutoff to obtain a filtrate, wherein the first cutoff is greater than the molecular weight of the peptides in the first peptide group and the first cutoff is less than the molecular weight of the antibody.

[0057] S3. Performing enzymatic hydrolysis on the filtrate by using trypsin, and terminating the reaction after a second period of time to obtain a test sample.

[0058] Step S1 includes:

[0059] S101. Add 50 mmol / L Tris-HCl buffer to 1 mg of product sample to obtain a total volume of 100 μL of solution, then add 20 μg of trypsin and incubate at 37°C for 3 h to obtain a preliminary enzymatic solution.

[0060] S102. Add horse cytochrome C, horse myoglobin, bovine β-lactoglobulin, bovine serum albumin, β-casein and chicken lysozyme in a mass ratio of 100:50:25:5:1:0.4 to the preliminary enzymatic hydrolysis solution as marker proteins, and the total concentration of the marker proteins is 3.2 ppm.

[0061] Step S2 comprises: ultrafiltration with an ultrafiltration tube having a cutoff of 100 kD. The centrifugal conditions for each ultrafiltration are 12000 g, 4° C., and 30 min. After each ultrafiltration, 100 μL of 50 mmol / L Tris-HCl is added for the next ultrafiltration, and ultrafiltration is performed three times in total to obtain a filtrate.

[0062] Step S3 includes:

[0063] S301. After ultrafiltration, 20 μg of trypsin was added and incubated at 37° C. for 20 h. After incubation, formic acid was added to make the final concentration of formic acid 0.1%, thereby terminating the reaction.

[0064] S302. Desalting is then performed using a mixed cation solid phase extraction column (MCX), and the desalted sample is freeze-dried and re-dissolved with 0.1% formic acid to a protein concentration of 1 μg / μl to obtain a test sample.

[0065] See also Figure 2 , the method for detecting host cell proteins in a product sample by using the above-mentioned test sample includes:

[0066] S4. Detecting the protein types and corresponding detection response values ​​in the test sample prepared by the above-mentioned preparation method by liquid chromatography-mass spectrometry.

[0067] S5. Prepare multiple external standard samples by the same method as in S4 to obtain multiple external standard detection samples, and detect the external standard response values ​​of the multiple external standard detection samples by liquid chromatography-mass spectrometry, and draw external standard curves. The multiple external standard detection samples respectively include labeled peptides with different concentrations, and the labeled peptides are obtained by hydrolyzing labeled proteins with different concentrations. The molecular weight of the labeled peptides is less than the first retention amount, and the host cell proteins do not contain labeled proteins.

[0068] S6. Determine the detection concentration based on the detection response value and the external standard curve.

[0069] Step S4 includes: testing the above 6 test samples respectively through LC-MS, performing data analysis according to the test results, and obtaining the protein types in the test samples and the corresponding test response values.

[0070] Step S5 includes:

[0071] S501. Take 1 mg from each of the 6 product samples and mix them to obtain a mixed sample, and take 1 mg from each of the mixed samples to obtain 4 external standard samples.

[0072] S502, repeat step S101 to process the external standard sample to obtain 4 preliminary enzymatic hydrolysates.

[0073] S503. Different masses of the same labeled protein as in step S102 are added to the four preliminary enzymatic hydrolysates, and the masses added to each preliminary enzymatic hydrolysate are 0.5 μg, 2.5 μg, 5 μg and 10 μg, respectively.

[0074] S504, repeat steps S2, S3 and S4 to obtain the external standard response value corresponding to each protein type in the labeled protein in each external standard sample, and draw an external standard curve of the external standard response value and the concentration value, see Figure 3 In this embodiment, the total protein concentration is used as the concentration value, and the total peak area is used as the external standard response value.

[0075] Step S6 includes:

[0076] The type of protein and the corresponding detection response value obtained in step S4 are combined with the external standard curve obtained in step S5 to obtain the type and corresponding concentration of the host cell protein in the product sample.

[0077] The total HCP concentration in this example is shown in Table 1 below.

[0078] Table 1:

[0079] sample Number of proteins identified Total protein content (ppm) AC 122 1040.47 VIN 90 663.88 IDF 4 35.40 AEX 3 22.92 CEX 2 19.47 UF 3 3.86

[0080] To verify the reliability of the test data in this example, the total HCP concentration of 6 product samples was tested using an ELISA kit purchased from Shanghai Duoning Biotechnology Co., Ltd. The test results are shown in Table 2 below.

[0081] Table 2:

[0082] sample Total protein content (ppm) AC 1091 VIN 778 IDF 13 AEX 10 CEX 6 UF 4

[0083] Comparing the data in Table 1 and Table 2, taking into account that the concentration of HCP is at the ppm level, and that the HCP content in the product samples taken from the same sample fluctuates at the ppm level, the difference between the data in Table 1 and Table 2 is within a reasonable range, so the detection scheme in this embodiment has a higher accuracy.

[0084] Embodiment 2:

[0085] The difference between this embodiment and the first embodiment is that this embodiment does not include step S102, and step S5 of this embodiment only includes:

[0086] Using labeled proteins of different masses as external standard samples, repeat steps S1 (excluding step S102), S2, S3 and S4 to obtain external standard response values ​​corresponding to each protein type in each external standard sample, and draw external standard curves of external standard response values ​​and concentration values.

[0087] Although the accuracy of the detection value determined by this method is slightly reduced due to different matrix effects, the degree of reduction is within an acceptable range. In addition, the detection method is simple, the standard curve can be used multiple times, the cost is low, and it has high practicality.

[0088] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a test sample for host cell protein residues, characterized in that: include: Performing an enzymatic hydrolysis treatment on a product sample containing host cell proteins and antibodies by trypsin for a first period of time to obtain a preliminary enzymatic hydrolyzate, wherein the preliminary enzymatic hydrolyzate includes a first peptide group obtained by enzymatic hydrolysis of the host cell protein and the antibody; Ultrafiltration treatment of the preliminary enzymatic hydrolysate to obtain a filtrate, wherein the cutoff of the ultrafiltration treatment is a first cutoff, the first cutoff is greater than the molecular weight of the peptides in the first peptide group, and the first cutoff is less than the molecular weight of the antibody; The filtrate is enzymatically treated with trypsin, and the reaction is terminated after a second period of time to obtain the test sample.

2. The preparation method according to claim 1, characterized in that The first time is any value between 2.5 h and 3.5 h, and the first retention amount is any value between 50 kD and 100 kD.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the trypsin used to treat the product sample to the product sample is 1: any value in the range of (30-100), and the mass ratio of the trypsin used to treat the filtrate to the product sample is 1: any value in the range of (30-100).

4. The preparation method according to claim 2, characterized in that: The ultrafilter is an ultrafiltration tube, and filtering with the ultrafiltration tube includes centrifugation at a speed of any value between 10000g and 15000g for a time of any value between 20min and 40min.

5. The preparation method according to claim 1, characterized in that: The filtrate is subjected to enzymatic hydrolysis by the trypsin and the reaction is terminated, followed by desalting.

6. A method for detecting host cell protein residues, characterized in that: include: Detecting the protein types and corresponding detection response values ​​in the test sample prepared by the preparation method according to any one of claims 1 to 5 by liquid chromatography-mass spectrometry; A plurality of external standard samples are prepared by the same method as the preparation method of the test sample to obtain a plurality of external standard test samples, and the external standard response values ​​of the plurality of external standard test samples are detected by the liquid chromatography-mass spectrometry, and an external standard curve is drawn, wherein the plurality of external standard test samples respectively include labeled peptide segments with different concentrations, and the labeled peptide segments are obtained by hydrolyzing labeled proteins with different concentrations, the molecular weight of the labeled peptide segment is less than the first retention amount, and the host cell protein does not contain the labeled protein; The detection concentration is obtained according to the detection response value and the external standard curve.

7. The detection method according to claim 6, characterized in that The marker protein includes one or more of horse cytochrome C, horse myoglobin, bovine β-lactoglobulin, bovine serum albumin, β-casein, and chicken lysozyme.

8. The detection method according to claim 6, characterized in that The labeled protein is quantitatively added to the preliminary enzymatic hydrolysis solution, and the external standard sample is obtained by mixing a plurality of the product samples.

9. The detection method according to claim 6, characterized in that: The external standard samples are the labeled proteins of different masses.

10. The detection method according to any one of claims 6 to 9, characterized in that: The test sample is freeze-dried and then redissolved in an organic solvent of a mobile phase in the liquid chromatography-mass spectrometry detection, and then the liquid chromatography-mass spectrometry detection is performed.

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