Method for determining content of donemab in blood sample by adopting liquid chromatography-tandem mass spectrometry
By combining enzymatic hydrolysis and internal standard method with liquid chromatography-tandem mass spectrometry detection, characteristic peptides were screened and mass spectrometry conditions were optimized, which solved the sensitivity and accuracy problems of donezumab drug detection, achieved efficient monitoring of donezumab concentration, and met clinical medication needs.
Patent Information
- Application Number
- CN202510843033.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-09-09
AI Technical Summary
The existing technology lacks sensitive, accurate and rapid liquid chromatography-tandem mass spectrometry detection methods for the quantitative and qualitative analysis of donezumab, which cannot meet the clinical drug testing needs of donezumab.
Serum samples were enzymatically treated to obtain characteristic peptides. Combined with the internal standard method and liquid chromatography-tandem mass spectrometry detection, the quantitative and qualitative analysis of donetumab was achieved by screening the characteristic peptides of donetumab and optimizing the mass spectrometry detection conditions.
A method for detecting the concentration of donezumab with high sensitivity, good specificity, high accuracy and good repeatability is provided, which can accurately monitor the content of donezumab in serum and guide clinical medication.
Smart Images
Figure CN120609935A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug detection and analysis, and particularly relates to a method for determining the content of donezumab in a blood sample by adopting liquid chromatography tandem mass spectrometry. Background Art
[0002] Donanemab is a humanized IgG1 monoclonal antibody therapeutic for Alzheimer's disease (AD) that was launched in China in March 2025. It is used to treat early AD. This donemab drug exerts its therapeutic effect by clearing β-amyloid protein (Aβ) plaques and can slow the rate of decline in cognitive function in patients with early Alzheimer's disease.
[0003] The current technology for detecting monoclonal antibody drug concentrations in human blood samples (blood drug concentration) mainly uses methods based on antigen-antibody immune reactions, such as enzyme-linked immunosorbent assay (ELISA), followed by liquid chromatography-tandem mass spectrometry. In comparison, liquid chromatography-tandem mass spectrometry offers higher sensitivity, better specificity, and better traceability, and is more capable of meeting the current clinical needs for drug concentration monitoring.
[0004] While liquid chromatography-tandem mass spectrometry (LC-MS) has excellent specificity, its operating conditions are also more demanding. For specific monoclonal antibodies, achieving precise and quantitative detection requires careful exploration of specific detection procedures.
[0005] Currently, there is no literature report on the use of liquid chromatography-tandem mass spectrometry for donesumab, a newly launched antibody drug.
[0006] Therefore, for the specific drug donezumab, there is an urgent need to establish a sensitive, accurate and rapid liquid chromatography-mass spectrometry detection method in this field to better meet the needs of clinical drug testing. Summary of the Invention
[0007] In order to solve the above problems, the present invention provides a method for determining the content of donezumab in a blood sample, which comprises enzymatically treating the serum sample to obtain a characteristic peptide segment, and determining the content of donezumab by liquid chromatography-tandem mass spectrometry detection in combination with an internal standard method; wherein,
[0008] The characteristic peptide segments generated after the donezumab is treated with enzymes include at least a first characteristic peptide segment for quantitative analysis, and the amino acid sequence of the first characteristic peptide segment is shown in SEQ ID NO: 1.
[0009] In one or more embodiments of the present application, the characteristic peptide segment produced after the enzymatic hydrolysis of donesumab also includes a second characteristic peptide segment and a third characteristic peptide segment for qualitative analysis; the amino acid sequences of the second and third characteristic peptide segments are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
[0010] In one or more embodiments of the present application, the internal standard method uses cetuximab as the internal standard.
[0011] Preferably, the sequence of the characteristic peptide generated by enzymatic hydrolysis of cetuximab is shown in SEQ ID NO: 4.
[0012] In one or more embodiments of the present application, the mass spectrometry detection conditions adopt multiple reaction monitoring (MRM) mode, positive ion mode, electrospray ionization, and an ion source temperature of 500-700°C.
[0013] Preferably, the mass spectrometry analysis parameters of the first characteristic peptide segment of donesumab include: the m / z value of the parent ion is 507.3, the m / z value of the daughter ion is 687.4, the DP value is 80.0V, and the CE value is 23.9V;
[0014] The mass spectrometry analysis parameters of the second characteristic peptide segment of donesumab include: the m / z value of the parent ion is 1114.5, the m / z value of the daughter ion is 660.3, the DP value is 80.0V, and the CE value is 53.6V;
[0015] The mass spectrometry analysis parameters of the third characteristic peptide segment of donesumab include: the m / z value of the parent ion is 429.7, the m / z value of the daughter ion is 630.3, the DP value is 80.0V, and the CE value is 20.1V;
[0016] The mass spectrometry analysis parameters of the characteristic peptide segment of cetuximab include: the m / z value of the parent ion is 597.0, the m / z value of the daughter ion is 652.0, the DP value is 80.0V, and the CE value is 15.0V.
[0017] In one or more embodiments of the present application, the mass spectrometry detection conditions include: ion source temperature of 600°C; electrospray voltage of 5500V; nebulizer gas of 65.0psi; auxiliary heating gas of 60.0°C; curtain gas of 30.0psi; and collision gas of 8psi.
[0018] In one or more embodiments of the present application, the liquid chromatography uses a C18 chromatographic column, the mobile phase A used is an aqueous solution containing 0.1% v / v formic acid, and the mobile phase B is an acetonitrile solution containing 0.1% v / v formic acid.
[0019] Gradient elution conditions were as follows:
[0020]
[0021]
[0022] Preferably, the flow rate is 0.2 to 0.4 mL / min.
[0023] In one or more embodiments of the present application, in the method, the process of enzymatically treating the serum sample to obtain characteristic peptide segments comprises the following steps:
[0024] Step 1), respectively preparing the standard solution and quality control solution of the donepezil monoclonal antibody, as well as the internal standard solution;
[0025] Step 2) adding the standard solution, the quality control solution, and the sample to be tested to corresponding amounts of Protein G magnetic beads, respectively; then adding the internal standard solution to each of them for enrichment and elution;
[0026] Step 3) The eluate obtained in step 2) is subjected to the enzymatic hydrolysis treatment to obtain the respective characteristic peptide segments; the enzymatic hydrolysis treatment conditions include first adding ammonium bicarbonate for denaturation, and then adding pancreatic enzyme solution for enzymatic hydrolysis.
[0027] In one or more embodiments of the present application, in step 1), the standard and control of donesumab are gradiently diluted using blank human serum to obtain standard solutions and control solutions of corresponding concentrations; preferably, the gradient concentrations of the standard solution are 0.5, 1, 5, 10, 50 and 200 μg / mL, respectively; the gradient concentrations of the control solution are 2, 20 and 100 μg / mL, respectively.
[0028] In one or more embodiments of the present application, in step 1), the internal standard solution is a cetuximab solution with a concentration of 100 μg / mL obtained by dilution with ultrapure water.
[0029] In one or more embodiments of the present application, in step 3), after the pancreatic enzyme solution is added, the working concentration of the pancreatic enzyme is 0.03-0.08 μg / mL.
[0030] The present invention relates to a method for determining the content of donezumab in a blood sample by using liquid chromatography tandem mass spectrometry; the method comprises subjecting a serum sample to enzymatic hydrolysis to obtain a characteristic peptide segment, and determining the content of the donezumab by a liquid chromatography tandem mass spectrometry detection method in combination with an internal standard method; the present application screens specific characteristic peptide segments of donezumab for quantitative and qualitative analysis, which can be used for determining the concentration of donezumab in serum, has high detection sensitivity, good specificity, high accuracy, and good repeatability, provides a reliable detection method for monitoring the concentration of donezumab, and can be used for guiding the clinical use of donezumab. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The liquid chromatography tandem mass spectrometry diagrams are of the characteristic peptides of donetumab and the internal standard cetuximab;
[0032] Figure 2 The standard curve graph of the characteristic peptide segment of donesumab (and the standard curve equation calculated from the graph);
[0033] Figure 3 Liquid chromatography tandem mass spectra of four samples. DETAILED DESCRIPTION
[0034] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to the scope of the examples. The process parameters not specified in the examples of this application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.
[0035] Example 1: Method for determining the content of donezumab in a blood sample
[0036] The method of the present application for determining the content of donezumab in a blood sample mainly involves enzymatic hydrolysis of a serum sample to obtain characteristic peptides, and then determining the content of donezumab in the blood sample by liquid chromatography-tandem mass spectrometry detection in combination with an internal standard method.
[0037] The method consists of two main stages: the first stage is enzymatic hydrolysis of serum samples to obtain characteristic peptides; the second stage is liquid chromatography-tandem mass spectrometry detection; the details are as follows.
[0038] 1. Phase 1
[0039] In some specific embodiments of the present application, the process of enzymatically treating a serum sample to obtain characteristic peptides mainly includes the following steps:
[0040] Step 1), respectively preparing the standard solution and quality control solution of the donepezil monoclonal antibody, as well as the internal standard solution;
[0041] Step 2) adding the standard solution, the quality control solution, and the sample to be tested to corresponding amounts of Protein G magnetic beads, respectively; then adding the internal standard solution to each of them for enrichment and elution;
[0042] Step 3) The eluate obtained in step 2) is subjected to the enzymatic hydrolysis treatment to obtain the respective characteristic peptide segments; the enzymatic hydrolysis treatment conditions include first adding ammonium bicarbonate for denaturation, and then adding pancreatic enzyme solution for enzymatic hydrolysis.
[0043] Regarding step 1), specifically in this embodiment 1, the operation is as follows:
[0044] 1-1) Preparation of Donetuzumab standard solution;
[0045] Donanemab, its English name is Donanemab (abbreviated DN), and its molecular formula is C 6452 H 10038 N 1708 O 2013 S 42 , CAS registration number 1931944-80-7.
[0046] The "original solution" for preparing the standard solution was prepared using clinically used donezumab injection purchased from Eli Lilly and Company, with a concentration of 17.5 mg / ml.
[0047] A blank matrix (blank human serum) was used for gradient dilution to obtain six standard solutions with gradient concentrations from low to high, namely 0.5, 1, 5, 10, 50 and 200 μg / mL. The six standard solutions were labeled as STD01, STD02, STD03, STD04, STD05 and STD06.
[0048] The preparation parameters of the six standard solutions are shown in Table 1 below:
[0049] Table 1
[0050]
[0051]
[0052] 1-2) Preparation of quality control solution of donezumab;
[0053] The "original solution" for preparing the quality control solution was prepared using clinically used donemab injection purchased from Eli Lilly and Company, with a concentration of 17.5 mg / ml.
[0054] A blank matrix (blank human serum) was used for gradient dilution to obtain three quality control solutions with gradient concentrations of 2, 20, and 100 μg / mL, respectively, labeled: LQC, MQC, and HQC. The preparation parameters of the three quality control solutions are shown in Table 2 below:
[0055] Table 2
[0056]
[0057] 1-3) Preparation of internal standard solution;
[0058] Regarding the selection criteria for the internal standard, first, it needs to have similar chemical and physical properties to "donezumab"; second, it must be able to remain stable during sample processing and detection without degradation or denaturation; third, the characteristic peptide or ion pair of the internal standard cannot overlap or interfere with the signals of the target monoclonal antibody or other matrix components.
[0059] Considering that donezumab is a humanized IgG1 monoclonal antibody, the inventors of the present application selected several monoclonal antibodies including trastuzumab, infliximab, cetuximab, and rituximab from known IgG1 monoclonal antibodies with similar properties for testing. They finally found that cetuximab was the most effective internal standard, with basically no interference, and could better avoid the problem of sample loss during the pretreatment process.
[0060] Cetuximab, its English name is Cetuximab (abbreviated as XTX), and its molecular formula is C 6484 H 10042 N 1732 O 2023 S 36 , CAS registration number 205923-56-4.
[0061] To prepare the internal standard solution, use clinically available cetuximab injection (Erbitux) purchased from Merck Pharmaceuticals (Jiangsu) Co., Ltd. at a concentration of 5 mg / mL. Dilute with ultrapure water to obtain a first-stage cetuximab working solution with a concentration of 1.00 mg / mL and store at -20°C until use. Accurately pipette an appropriate amount of the working solution and dilute with ultrapure water to prepare an internal standard solution with a concentration of 100 μg / mL.
[0062] Regarding step 2), specifically in this embodiment 1, the operation is as follows:
[0063] 2-a) According to the amount of the above-mentioned standard solution, quality control solution and blood sample to be tested, 40.0 μL of Protein G magnetic beads were taken and washed twice with 500 μL of PBS solution.
[0064] 2-b) Add 250 μL of PBS containing 0.05% TW20 to the washed magnetic beads, followed by 20.0 μL of standard solutions (STD01, STD02, STD03, STD04, STD05, and STD06), quality control solution, and the blood sample to be tested. Then, add 10.0 μL of internal standard solution (100 μg / mL cetuximab solution) to each sample and vortex at 1000 rpm to mix thoroughly.
[0065] 2-c) Incubate on a RM100 roller mixer for 1 h (roller speed 80), then wash twice with 500 μL of PBS solution containing 0.1% BSA and twice with deionized water.
[0066] 2-d) The monoclonal antibody was eluted from the magnetic beads using 100 μL of 0.25% FA solution and vortexed at 2000 rpm for 10 min; the eluate of each sample was collected.
[0067] Regarding the enzymatic hydrolysis treatment in step 3), specifically in this embodiment 1, the operation is as follows:
[0068] 3-1) denaturation;
[0069] Take 90 μL of the eluate of each sample obtained above, add 17 μL of 1 M ammonium bicarbonate solution, and denature at 95°C with shaking for 30 min;
[0070] 3-2) Enzymatic hydrolysis;
[0071] The denatured solution was cooled to room temperature, and 15 μL of 0.5 μg / μL pancreatin solution (the working concentration of pancreatin is approximately 0.0615 μg / μL) was added to the solution. The solution was vortexed at 600 rpm and placed at 37°C for 2 h (shaking at 600 rpm).
[0072] 23.3 μL of 10% formic acid in 50% acetonitrile was added to the enzymatic hydrolysate, vortex-mixed at 2000 rpm, and centrifuged at 15000 rpm for 5 min. The supernatant was collected for the second stage of liquid chromatography-tandem mass spectrometry detection described below.
[0073] Phase II: Liquid chromatography tandem mass spectrometry detection
[0074] 1. Liquid chromatography
[0075] For the specific "donezumab" targeted by this application, its liquid chromatography conditions need to be further optimized based on conventional operating parameters. The inventors of this application have optimized the liquid chromatography conditions suitable for "donezumab" through extensive experiments, especially the gradient elution conditions.
[0076] The conditions of liquid chromatography used in this embodiment are:
[0077] Column: Kinetex C18, 3.0 × 100 mm, particle size 2.6 μm;
[0078] Mobile phase A: water containing 0.1% formic acid;
[0079] Mobile phase B: acetonitrile containing 0.1% formic acid;
[0080] Needle wash solution: 50% methanol;
[0081] Gradient elution was used, the column temperature was 45°C, and 5 μL was injected. The gradient elution conditions are shown in Table 3 below:
[0082] Table 3
[0083]
[0084] The eluate obtained by liquid chromatography separation was sent to the following mass spectrometer for detection.
[0085] 2. Mass spectrometry
[0086] a) Mass spectrometry conditions
[0087] After experimental optimization by the inventors of this application, the mass spectrometry conditions are as follows:
[0088] The present invention uses an AB 6500+Citrine mass spectrometer (AB sciex, USA).
[0089] Multiple reaction monitoring (MRM) mode and positive ion mode were used, and the ion source used was electrospray ionization (ESI);
[0090] In this embodiment, the ion source temperature is 600° C.; the electrospray voltage is 5500 V; the nebulizer gas (Gas1) is 65.0 psi; the auxiliary heating gas (Gas2) is 60.0° C.; the curtain gas (Gurtain Gas) is 30.0 psi; and the collision gas (CAD) is 8 psi.
[0091] b) Characteristic peptides
[0092] The inventors of the present application matched the mass-to-charge ratios of the product ions obtained by mass spectrometry with the theoretical peptide sequences in the database and deduced the amino acid sequences of the peptides of donesumab and the internal standard monoclonal antibody;
[0093] Through blast comparison analysis, the specificity and specificity of the theoretical enzymatic peptides of donesumab and internal standard monoclonal antibody were determined, and specific peptides were screened out, including characteristic peptides that can be used for qualitative and quantitative analysis.
[0094] After multiple experiments and comparative analysis, the inventors of this application screened for characteristic peptides suitable for mass spectrometry detection of donezumab. Among them, the most important is the first characteristic peptide (shown in SEQ ID NO: 1) used for quantitative analysis, which can accurately detect the concentration of donezumab.
[0095] In addition to the characteristic peptide segment for quantitative analysis, the inventors of this application also screened out two more specific characteristic peptide segments for qualitative analysis: a second characteristic peptide segment (shown in SEQ ID NO: 2) and a third characteristic peptide segment (shown in SEQ ID NO: 3). The combination of the first characteristic peptide segment for quantitative analysis and the second and third characteristic peptide segments for qualitative analysis can more sensitively detect the concentration of donezumab, with a lower limit of detection (minimum concentration).
[0096] For the internal standard "cetuximab", the inventors of the present application screened out a characteristic peptide segment for its quantitative analysis, as shown in SEQ ID NO: 4.
[0097] Using the above mass spectrometry detection conditions, the mass spectrometry analysis parameters (MRM scanning parameters) of the above four characteristic peptides are shown in Table 4 below:
[0098] Table 4
[0099]
[0100] like Figure 1 As shown in the figure, under the detection conditions, the peak time of the first characteristic peptide segment (quantitative characteristic peptide segment) of donesumab is about 6.73 min, and the peak times of the second and third characteristic peptide segments (qualitative characteristic peptide segments) are 8.07 min and 2.50 min respectively; the peak time of the characteristic peptide segment of cetuximab is 5.56 min.
[0101] 3. Quantitative calculation
[0102] The standard solution after detection was used to draw the standard curve equation, with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio as the Y-axis, and linear regression analysis was performed to obtain the standard curve and the standard curve equation, as shown in the figure. Figure 2 shown.
[0103] Standard curve liquid chromatography tandem mass spectrometry Figure 3 shown.
[0104] After data analysis, the intra-batch precision, accuracy and inter-batch precision, accuracy results detected according to the method of this Example 1 are shown in Table 5 below.
[0105] Table 5
[0106]
[0107]
[0108] STD01-06 represent standard concentrations, LQC, MQC, and HQC represent low, medium, and high quality controls, respectively.
[0109] From the results in Table 5, it can be seen that the standard curve can be quantified as low as 0.5ug / mL using the measurement method of the present application. This result can also be seen from Figure 3 Be confirmed.
[0110] Figure 3 These are liquid chromatography tandem mass spectrometry graphs of four samples, where the subgraph marked DB is the result graph of the double blank sample (no donezumab standard and no internal standard added), the subgraph marked STD00 is the result graph of the single blank sample (no donezumab standard added, but internal standard added), the subgraph marked 0.5ug / mL is the result graph of the donezumab standard solution with a concentration of 0.5ug / mL, and the subgraph marked 1ug / mL is the result graph of the donezumab standard solution with a concentration of 1ug / mL.
[0111] from Figure 3 It can also be seen that the background of the tandem chromatography-mass spectra of the double blank and single blank samples is clean, and there is no obvious interference at the peaks of the characteristic peptide segments; however, when the concentration of donezumab reaches above 0.5 ug / mL, the response of donezumab is high and clearly separated from the baseline, indicating that the measurement method of the present application can be used to quantitatively analyze donezumab in serum, and the minimum concentration that can be detected can be as low as 0.5 ug / mL, with high detection sensitivity.
[0112] In addition, it can be seen from the results in Table 5 that the average recovery rates of low, medium and high quality control products in the measurement method of the present application are all around 100%, and the intra-batch and inter-batch CV% are all less than 10%, indicating that the measurement method of the present application has high accuracy and good repeatability.
[0113] The present invention is not limited to the above-described embodiments. The scope of protection of the present invention is defined by the appended claims. Those skilled in the art may make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of the present invention. All of these changes, modifications, substitutions, combinations, and simplifications are equivalent and fall within the scope of protection of the present invention.
Claims
1. A method for determining the content of donezumab in a blood sample, the method comprising enzymatically hydrolyzing a serum sample to obtain a characteristic peptide segment, and determining the content of donezumab by liquid chromatography-tandem mass spectrometry detection in combination with an internal standard method; wherein, The characteristic peptide segments generated after the donezumab is treated with enzymes include at least a first characteristic peptide segment for quantitative analysis, and the amino acid sequence of the first characteristic peptide segment is shown in SEQ ID NO:
1.
2. The method according to claim 1, wherein: The characteristic peptide segments produced after enzymatic hydrolysis of the donezumab also include a second characteristic peptide segment and a third characteristic peptide segment for qualitative analysis; the amino acid sequences of the second and third characteristic peptide segments are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
3. The method according to claim 1 or 2, wherein: The internal standard method uses cetuximab as the internal standard; Preferably, the sequence of the characteristic peptide generated by enzymatic hydrolysis of cetuximab is shown in SEQ ID NO:
4.
4. The method according to claim 3, wherein: The mass spectrometry detection conditions adopted multiple reaction monitoring mode, positive ion mode, electrospray ionization, and ion source temperature of 500-700°C; Preferably, the mass spectrometry analysis parameters of the first characteristic peptide segment of donesumab include: the m / z value of the parent ion is 507.3, the m / z value of the daughter ion is 687.4, the DP value is 80.0V, and the CE value is 23.9V; The mass spectrometry analysis parameters of the second characteristic peptide segment of donesumab include: the m / z value of the parent ion is 1114.5, the m / z value of the daughter ion is 660.3, the DP value is 80.0V, and the CE value is 53.6V; The mass spectrometry analysis parameters of the third characteristic peptide segment of donesumab include: the m / z value of the parent ion is 429.7, the m / z value of the daughter ion is 630.3, the DP value is 80.0V, and the CE value is 20.1V; The mass spectrometry analysis parameters of the characteristic peptide segment of cetuximab include: the m / z value of the parent ion is 597.0, the m / z value of the daughter ion is 652.0, the DP value is 80.0V, and the CE value is 15.0V.
5. The method according to claim 4, wherein: The mass spectrometry detection conditions include: ion source temperature of 600°C; electrospray voltage of 5500V; nebulizer gas of 65.0psi; auxiliary heating gas of 60.0°C; curtain gas of 30.0psi; The collision gas was 8 psi.
6. The method according to claim 3, wherein: The liquid chromatography used a C18 chromatographic column, the mobile phase A used was an aqueous solution containing 0.1% v / v formic acid, and the mobile phase B used was an acetonitrile solution containing 0.1% v / v formic acid. Gradient elution conditions were as follows: Preferably, the flow rate is 0.2 to 0.4 mL / min.
7. The method according to any one of claims 3 to 6, characterized in that: In the method, the process of enzymatically treating the serum sample to obtain characteristic peptide segments comprises the following steps: Step 1), respectively preparing the standard solution and quality control solution of the donepezil monoclonal antibody, as well as the internal standard solution; Step 2) adding the standard solution, the quality control solution, and the sample to be tested to corresponding amounts of Protein G magnetic beads, respectively; then adding the internal standard solution to each of them for enrichment and elution; Step 3) The eluate obtained in step 2) is subjected to the enzymatic hydrolysis treatment to obtain the respective characteristic peptide segments; the enzymatic hydrolysis treatment conditions include first adding ammonium bicarbonate for denaturation, and then adding pancreatic enzyme solution for enzymatic hydrolysis.
8. The method according to claim 7, wherein: In the step 1), the standard and control of the donezumab are gradiently diluted using blank human serum to obtain standard solutions and control solutions of corresponding concentrations; preferably, the gradient concentrations of the standard solutions are 0.5, 1, 5, 10, 50 and 200 μg / mL, respectively; the gradient concentrations of the control solutions are 2, 20 and 100 μg / mL, respectively.
9. The method according to claim 7, wherein: In the step 1), the internal standard solution is a cetuximab solution with a concentration of 100 μg / mL obtained by diluting with ultrapure water.
10. The method according to claim 7, wherein: In step 3), after adding the pancreatic enzyme solution, the working concentration of the pancreatic enzyme is 0.03-0.08 μg / mL.