Method for simultaneously determining various saccharide freeze-drying protective agents in monoclonal antibody medicine

Abstract

The invention belongs to the technical field of biopharmacy analysis, and particularly relates to a method for simultaneously determining various saccharide freeze-drying protective agents in monoclonal antibody drugs. The test solution is detected based on the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technology, the sample pretreatment process, chromatographic separation conditions and mass spectrometry detection parameters are optimized, and trehalose and cane sugar in the monoclonal antibody preparation are rapidly, sensitively and accurately measured. The method provided by the invention solves the problems that the existing high performance liquid chromatography method can only singly detect trehalose or sucrose in the monoclonal antibody and the analysis efficiency is low, overcomes the problem of cross interference of mass spectrum signals, and ensures the accuracy of target object quantification. In addition, by optimizing pretreatment, the problems of matrix effect and solvent effect are solved. The method has the advantages of high sensitivity, high accuracy and good repeatability, and provides an innovative solution for quality control and stability research of monoclonal antibody drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical analysis, and particularly relates to a method for simultaneously determining multiple saccharide lyoprotectants in monoclonal antibody drugs. Background Art

[0002] Monoclonal antibodies (mAbs), as an important biopharmaceutical product, play a crucial role in the treatment of various refractory diseases due to their high specificity and efficiency. During the production and storage of mAbs, lyophilization is a commonly used technique to improve the stability of the drug and extend its shelf life. However, the lyophilization process may cause stress on the protein structure, leading to denaturation, aggregation, or degradation. To overcome this problem, saccharide protectants are widely added to mAb formulations to stabilize the protein structure during lyophilization and storage, preventing its inactivation. Among them, sucrose and trehalose are the most commonly used saccharide lyoprotectants in mAb drugs, which can effectively prevent protein aggregation during freeze-drying and storage through mechanisms such as forming a glassy matrix, replacing water molecules, and reducing phase change stress. However, excessive use of trehalose and sucrose may trigger new quality risks, such as increased drug viscosity, adverse reactions at the injection site, and even interference with the binding activity of mAbs to their targets, affecting the stability, function, and safety of mAbs. Therefore, in the development and production of mAb formulations, reasonable control of the dosage of trehalose and sucrose is crucial for ensuring the quality and safety of mAb drugs.

[0003] Currently, the quantitative analysis methods for trehalose and sucrose in mAb drugs are mainly based on high-performance liquid chromatography (HPLC) technology, combined with different detectors and chromatographic columns to achieve the determination of these saccharide excipients. However, the existing HPLC methods can only achieve the single detection of trehalose or sucrose in mAbs, unable to simultaneously determine the two saccharide protectants, resulting in low analysis efficiency and resource waste, and there are still some limitations in terms of anti-interference ability. Given the limitations of the existing analysis methods, developing a rapid, sensitive, and reliable analysis method for determining the content of commonly used saccharide lyoprotectants in mAb drugs is of great significance for the quality control and stability research of mAb drugs. Summary of the Invention

[0004] In response to the above needs, the present invention provides a rapid, sensitive, and accurate analysis method for simultaneously determining the content of multiple commonly used saccharide lyoprotectants (such as trehalose and sucrose) in monoclonal antibody (mAb) drugs. This method can overcome the limitations of existing analysis techniques (such as high-performance liquid chromatography) in terms of detection throughput and anti-interference ability, providing an innovative solution for the quality control and stability research of mAb drugs.

[0005] A method for simultaneously determining multiple saccharide cryoprotectants in monoclonal antibody drugs according to the present invention is to purify and dilute the test sample by solid-phase microextraction to obtain a test sample solution, and then detect it by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry.

[0006] The preparation of the test sample solution includes the following steps: (1) Dilute the test sample with deionized water to obtain a diluted test sample solution; (2) Activate the SPE column with methanol and equilibrate it with pure water. Then load the diluted test sample solution onto the equilibrated SPE column, collect the filtrate under positive pressure, wash it with pure water, and collect the filtrate; (3) Dilute the filtrate with acetonitrile to obtain a test sample solution; In step (1), the test sample is diluted 50,000 times with deionized water; In step (2), the SPE consumable used is Oasis PRiME HLB 96-well µElution Plate, 3 mg; In step (3), the volume ratio of the filtrate to acetonitrile is 1:1.

[0007] After preparing the test sample, analyze and detect it using a high performance liquid chromatography-tandem mass spectrometer.

[0008] The liquid chromatography conditions are as follows: ACQUITY UPLC Glycan BEH Amide Column, 150 mm×2.1 mm, 1.7 μm, column temperature 50 - 60 °C, mobile phase A is 0.1% - 0.2% ammonia water solution, mobile phase B is acetonitrile, gradient elution, injection volume is 2.0 μL; the gradient elution program is: 0.00 - 10.00 min, 85%B - 65%B, 10.01 - 11.00 min, 65%B - 50%B, 11.01 - 15.00 min, 85%B.

[0009] The mass spectrometry conditions are as follows: electrospray ionization source negative ion mode, desolvation tube temperature is 200 - 300 °C, heating module temperature is 400 °C, interface temperature is 300 °C, nebulizing gas is nitrogen, flow rate is 3.0 L / min, drying gas is nitrogen, heating gas is air, flow rates are all 10.0 L / min, the mass spectrometry detector detection mode is multiple reaction monitoring (MRM), and the MRM parameter table is shown in Table 1.

[0010] Table 1. MRM parameter table .

[0011] * is the quantitative ion.

[0012] The present invention also includes the preparation of a mixed standard working solution, and the specific steps are as follows: Weigh an appropriate amount of trehalose and sucrose precisely, dissolve them in deionized water to a concentration of 1 mg / mL, and gradually dilute them with an acetonitrile - aqueous solution (5:5) to a series of mixed standard working solutions with mass concentrations of 2, 5, 10, 50, 100, 200, and 500 ng / mL for determination by high - performance liquid chromatography - tandem mass spectrometry.

[0013] The saccharide lyophilization protectants described in the present invention are trehalose and sucrose. In the research and development of monoclonal antibody preparations, common disaccharides may include maltose, lactose, sucrose, and trehalose. Among them, maltose and lactose are often used as ion - strength - adjusting excipients in products, while trehalose and sucrose are mostly used as lyophilizing agents. Because of their similar molecular structures and the same molecular weight, they are likely to produce the same ion channels in mass spectrometry detection, resulting in deviations in quantitative results. Therefore, before the sample enters the mass spectrometer, it is necessary to ensure the full separation of trehalose and sucrose through chromatographic separation techniques. At the same time, it is also necessary to separate them from their respective isomers to avoid mutual interference during mass spectrometry detection and ensure the accuracy of the detection results.

[0014] The applicant has tried amino columns with silica gel matrices from different manufacturers. The amino group can retain saccharide molecules through hydrophilic interaction, but its silica - amino bonded phase is prone to hydrolysis in the aqueous mobile phase, resulting in the shedding of amino functional groups. The shed amino groups enter the mass spectrometry ion source and form adducts in electrospray ionization (ESI), leading to a decrease in sample signal and an increase in baseline noise. Using a polymer - matrix chromatographic column (Asahipak NH2P - 50 2D, 150 mm×2.0 mm, 5μm), it was found that it was difficult to separate trehalose and lactose by adjusting the mobile phase. Then, an amide column with a hybrid - particle silica gel matrix (ACQUITYUPLC Glycan BEH Amide) was considered for separation. Among them, maltose and lactose are reducing sugars, and reducing sugars will undergo mutarotation, generating α - type and β - type anomers. High temperature and high pH conditions can make the two isomers become one peak.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) Through the pretreatment strategy of SPME purification, the present invention successfully solves the problem of matrix effect in the traditional protein precipitation method (organic - phase precipitation - high - speed centrifugation), and further solves the solvent effect through acetonitrile dilution, providing a reliable solution for the high - precision quantitative analysis of saccharide protectants in monoclonal antibody preparations.

[0016] (2) By optimizing the chromatographic conditions, the present invention has successfully increased the resolution between trehalose, sucrose and their isomers (maltose, lactose) to ≥1.5, completely solving the problem of cross-interference of mass spectrometry signals caused by the same molecular weight and overlapping ion channels, thus avoiding false positive results and ensuring the accuracy of the quantification of the target substance.

[0017] (3) The present invention has established for the first time a detection method for saccharide lyophilization protectants in monoclonal antibody drugs based on LC-MS / MS coupling technology, providing an innovative analytical means for the quality control and stability research of monoclonal antibody drugs, which can be widely applied to the improvement of the quality control system in the biopharmaceutical industry and the management of the entire product life cycle.

[0018] (4) The detection limit of the method described in the present invention is between 0.20 ng / mL - 0.45 ng / mL, the quantification limit is between 0.67 ng / mL - 1.51 ng / mL, and the recovery rate of blank matrix spiking is between 91.3% - 98.5%. This method is sensitive and accurate and can be used for the simultaneous determination of common saccharide lyophilization protectants in monoclonal antibodies. Description of the Drawings

[0019] Figure 1 Chromatogram of the mixed standard solution of sucrose, maltose, lactose, and trehalose in the MRM channels of trehalose and sucrose under the column temperature condition of 50°C; Figure 2 Chromatogram of the mixed standard solution of sucrose, maltose, lactose, and trehalose in the multiple reaction monitoring (MRM) channels of trehalose and sucrose under the condition of phase A with different concentrations of ammonia water; Figure 3 Chromatogram of the mixed standard solution of sucrose (500 ng / mL), maltose (500 ng / mL), lactose (500 ng / mL), and trehalose (5000 ng / mL) in the MRM channels of trehalose and sucrose; Figure 4 Chromatogram of the multiple reaction monitoring (MRM) channel of trehalose in the mixed standard solution of 100 ng / mL of trehalose and sucrose at different desolvation tube (DL tube) temperatures; Figure 5 Chromatogram of the multiple reaction monitoring (MRM) channel of sucrose in the mixed standard solution of 100 ng / mL of trehalose and sucrose at different desolvation tube (DL tube) temperatures; Figure 6 Chromatogram of the mixed standard solution of 50 ng / mL of trehalose and sucrose; Figure 7 Chromatogram of the determination of actual sample A; Figure 8 Chromatogram of the determination of actual sample H; Figure 9Chromatogram measured for actual sample B; Figure 10 Overlay diagram measured for specificity experiment. Detailed implementation manner

[0020] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0021] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0022] Example 1 1.1. Experimental instruments and equipment High-pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

[0023] 1.2. Experimental reagents Acetonitrile and ammonia water are of mass spectrometry purity; water is Wahaha pure water; reference substances trehalose (CAS No. 99-20-7), sucrose (CAS No. 57-50-1), maltose (CAS No. 69-79-4), lactose (CAS No. 57-50-1).

[0024] 1.3. Detection conditions Liquid chromatography conditions: Chromatographic column: ACQUITY UPLC Glycan BEH Amide Column (150 mm×2.1 mm I.D., 1.7μm); Mobile phase: A - ammonia water: water (1 - 2:1000, v / v); B - acetonitrile; Gradient: 0 - 10.00 min 85%B - 65%B, 10.01 - 11.00 min 65%B - 50%B, 11.01 - 15.00 min 85%B, column temperature: 50 - 60 °C; flow rate: 0.25 mL / min; injection volume: 2.0 µL; Mass spectrometry conditions: Ion source: electrospray ionization source in negative ion mode; mass spectrometer: triple quadrupole liquid chromatography-mass spectrometry; desolvation tube temperature is 200 - 300 °C; heating module temperature is 400 °C; interface temperature is 300 °C; nebulizing gas is nitrogen with a flow rate of 3.0 L / min; drying gas is nitrogen, heating gas is air, and the flow rates are both 10.0 L / min; the detection mode of the mass spectrometry detector is multiple reaction monitoring (MRM), and the mass spectrometry detection parameters are shown in Table 1.

[0025] 1.4. Standard curve drawing 1) Preparation of standard stock solution: Weigh appropriate amounts of trehalose, sucrose, maltose, and lactose precisely, dissolve them in deionized water to form original stock solutions with a concentration of 100 mg / mL each, and then dilute them with deionized water to 1 mg / mL standard stock solutions for standby. 2) Preparation of standard working solutions: Accurately pipette appropriate amounts of the standard stock solutions of 2 sugars (trehalose, sucrose) respectively, and gradually dilute them with acetonitrile - aqueous solution (5:5) to a series of mixed standard working solutions with mass concentrations of 2, 5, 10, 50, 100, 200, and 500 ng / mL for determination by liquid chromatography - mass spectrometry. Among them, the chromatogram of the 50 ng / mL trehalose and sucrose mixed standard solution is shown in Figure 6 ; 3) Drawing of the standard working curve: Analyze the above standard working solutions by high performance liquid chromatography tandem mass spectrometry to obtain the chromatograms of the above reference substances, and obtain the standard working curve according to the relationship between the solution concentration and the corresponding chromatographic peak area.

[0026] 1.5. Preparation of test sample solution (1) Dilute the test sample 50,000 times with deionized water to obtain the diluted test sample solution; (2) Activate the SPE column with 500 μL of methanol, discard the filtrate after positive pressure; add 500 μL of pure water for equilibration, discard the filtrate after positive pressure; then take 500 μL of the diluted test sample solution and load it onto the equilibrated SPE column, collect the filtrate after positive pressure, and then add 500 μL of pure water for rinsing, collect the filtrate after positive pressure; (3) Take 500 μL of the filtrate and dilute it with 500 μL of acetonitrile to obtain the test sample solution; The SPE consumables used in this invention are Oasis PRiME HLB 96-well µElution Plate, 3 mg.

[0027] 1.6. Determination of the optimal detection conditions In the liquid chromatography conditions, when the column temperature is 50 °C, baseline separation of trehalose, sucrose, and their isomers can be achieved, as shown in Figure 1 shown; when the column temperature is increased to 60 °C, while ensuring the separation effect, the analysis efficiency is significantly improved, and it only takes 15 minutes to accurately quantify trehalose and sucrose in the sample.

[0028] The present invention uses ammonia water to adjust the mobile phase to a high pH value condition. During the optimization of mobile phase A, 0.2% ammonia water solution and 0.1% ammonia water solution were tried, and the chromatographic behaviors of the compounds to be measured were similar, as Figure 2 shown. In order to extend the service life of the chromatographic column, mobile phase A was finally determined to be 0.1% ammonia water solution. Under the optimized chromatographic conditions, a mixed standard solution of sucrose, maltose, lactose and trehalose was analyzed, and the results showed ( Figure 3 ): In the multiple reaction monitoring (MRM) channels of sucrose and trehalose, maltose and lactose also had signal responses. Their chromatographic peak retention times were highly close, overlapping into one chromatographic peak. The resolution between the chromatographic peaks of sucrose and trehalose and the two components of maltose and lactose both reached more than 1.5, successfully realizing the effective separation of trehalose, sucrose and their isomers. The obtained chromatographic peak baseline was stable, the peak shape was symmetric and sharp, significantly reducing the signal interference in mass spectrometry detection, thus effectively improving the accuracy and reliability of mass spectrometry quantitative analysis.

[0029] During the optimization of mass spectrometry conditions, the temperature of the desolvation tube (DL tube) had a significant impact on the response of the compounds. Therefore, the responses of a 100 ng / mL mixed standard solution of trehalose and sucrose at different desolvation tube temperatures (200 °C, 250 °C and 300 °C) were investigated, and the chromatograms are shown in Figures 4 - 5 . The results showed that under different desolvation tube (DL tube) temperature conditions, the response values of trehalose had little difference, while the response value of sucrose increased significantly when the desolvation tube temperature was 250 °C.

[0030] Therefore, the optimal detection conditions are as follows: Liquid chromatography conditions: Chromatographic column: ACQUITY UPLC Glycan BEH Amide Column (150 mm×2.1 mm I.D., 1.7 μm); Mobile phase: A - ammonia water: water (1:1000, v / v); B - acetonitrile; Gradient: 0 - 10.00 min 85%B - 65%B, 10.01 - 11.00 min 65%B - 50%B, 11.01 - 15.00 min 85%B, column temperature: 60 °C; flow rate: 0.25 mL / min; injection volume: 2.0 μL; Mass spectrometry conditions: Ion source: electrospray ionization source, negative ion mode; mass spectrometer: triple quadrupole liquid chromatography-mass spectrometry; desolvation tube temperature: 250 °C; heating module temperature: 400 °C; interface temperature: 300 °C; nebulizing gas: nitrogen, flow rate: 3.0 L / min; drying gas: nitrogen, heating gas: air, flow rates are both 10.0 L / min; mass spectrometry detector detection mode: multiple reaction monitoring (MRM), mass spectrometry detection parameters are shown in Table 1.

[0031] Example 2 Take 3 portions each of 3 commercially available monoclonal antibody drugs (Sample A: bevacizumab, Avastin, Roche; Sample H: trastuzumab, Herceptin, Roche; Sample B: belimumab, Benlysta, GSK), and prepare test solution according to the method under item "1.5" in Example 1 respectively, and conduct determination according to the chromatographic conditions under item "1.6" in Example 1. The chromatograms of Sample A, H, and B are respectively as Figure 7 , Figure 8 , Figure 9 shown. Substitute the peak areas of trehalose and sucrose in the sample into the fitting function to calculate the contents of trehalose and sucrose and their RSDs. The determination results of the actual samples are shown in Table 2.

[0032] Table 2. Determination results of actual samples (n = 3) .

[0033] Note: "N.D." means not detected, and " / " means not calculable.

[0034] Example 3 Specificity experiment Since the monoclonal antibody active ingredient in the monoclonal antibody sample to be tested has been purified by SPME and retained in the C18 reversed-phase column, the specificity verification solutions are 3 kinds in total, namely blank solution, preparation buffer without trehalose and sucrose, and a mixed reference solution of 10 ng / mL. Among them, take ultrapure water as the blank solution; configure a preparation buffer without trehalose and sucrose by taking 0.495 mg / mL histidine hydrochloride, 0.32 mg / mL histidine, 0.09 mg / mL polysorbate 20, 4.8 mg / mL sodium dihydrogen phosphate monohydrate, and 1.2 mg / mL disodium hydrogen phosphate. The results are as Figure 10 shown. At the retention time of trehalose at 8.745 min and the retention time of sucrose at 7.402 min, no interfering peaks were detected in the 3 specificity verification solutions, indicating that the monoclonal antibody drug itself and other substances in the preparation buffer do not interfere with the detection of trehalose. The results show that this method has high specificity.

[0035] Example 4 Sensitivity investigation Perform linear regression on the above standard working curve and investigate the method sensitivity. The correlation coefficients of the obtained calibration curves are all greater than 0.999, and the concentration read-back values of each calibration point are within the range of 95.9% - 104.7% of their theoretical concentrations. Analyze using the mixed standard solution at the lowest concentration point, and calculate its detection limit (S / N = 3) and quantification limit (S / N = 10) based on the signal-to-noise ratio. The linear equations, detection limits, and quantification limits are shown in Table 3. The results show that each reference substance has a good linear relationship within its respective mass range, proving that the method of the present invention has high accuracy and high method sensitivity within the selected linear range.

[0036] Table 3. Linear equations, detection limits, and quantification limits 。

[0037] Example 5 Repeatability investigation The 100.0 ng / mL mixed standard working solution was continuously measured 6 times to investigate the repeatability of the instrument. The measurement results are shown in Table 4. The RSD of the retention time is between 0.037% and 0.095%, and the RSD of the peak area is between 0.50% and 1.52%. The method has good repeatability.

[0038] Table 4. Repeatability results of retention time and peak area (n = 6) 。

[0039] Example 6 Recovery investigation Take blank matrix (0.495 mg / mL histidine hydrochloride, 0.32 mg / mL histidine, 0.09 mg / mL polysorbate 20, 4.8 mg / mL sodium dihydrogen phosphate monohydrate, 1.2 mg / mL disodium hydrogen phosphate, 1.1% benzyl alcohol solution) and add standard solution at low (series 2), medium (series 4), and high (series 6) 3 concentration levels respectively, and perform the determination according to the method established above. Each concentration level has 3 parallel samples in duplicate, and calculate the spiked recovery rate and relative standard deviation (RSD). The results are shown in Table 5. The spiked recovery rates of the saccharide compounds to be measured are between 91.3% and 98.5%, and the RSDs are between 0.80% and 2.25%.

[0040] Table 5. Spiked recovery rate results (n = 3) 。

[0041] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs, characterized in that: The test solution was tested based on ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; The liquid chromatography conditions were: ACQUITY UPLC Glycan BEH Amide column, column temperature 50-60°C, mobile phase A 0.1%-0.2% ammonia solution, phase B acetonitrile, gradient elution, phase B concentration range 85%-50%; The mass spectrometry conditions were: electrospray ion source negative ion mode, desolvation tube temperature 200-300°C, and mass spectrometry detector detection mode multiple ion selection monitoring; The sugar freeze-drying protective agents are trehalose and sucrose.

2. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 1, characterized in that: The specifications of the chromatographic column are 150 mm×2.1 mm, 1.7 μm.

3. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 1, characterized in that: In the HPLC conditions, the injection volume was 2.0 µL.

4. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 1, characterized in that: In the mass spectrometry conditions, the heating module temperature was 400°C; the interface temperature was 300°C; the nebulizing gas was nitrogen with a flow rate of 3.0 L / min; the drying gas was nitrogen, and the heating gas was air, both with a flow rate of 10.0 L / min.

5. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 1, characterized in that: The gradient elution program is: 0.00-10.00 min, 85% B-65% B, 10.01-11.00 min, 65% B-50% B, 11.01-15.00 min, 85% B.

6. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 1, characterized in that: The preparation of the test solution comprises the following steps: (1) Dilute the test sample with deionized water to obtain the test sample dilution solution; (2) Activate the SPE column with methanol and balance it with pure water. Then load the sample dilution onto the balanced SPE column, rinse it with pure water, and collect the filtrate. (3) Dilute the filtrate with acetonitrile to obtain the test solution.

7. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 6, characterized in that: In step (1), the test sample was diluted 50,000 times with deionized water.

8. The method for simultaneously determining multiple carbohydrate lyoprotectants in monoclonal antibody drugs according to claim 6, characterized in that: In step (3), the volume ratio of the filtrate to acetonitrile is 1:1.

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