A method for simultaneously determining multiple saccharide cryoprotectants in monoclonal antibody drugs

Abstract

The present invention belongs to the technical field of biopharmaceutical analysis, and particularly relates to a method for simultaneously determining multiple saccharide cryoprotectants in monoclonal antibody drugs. The present invention detects the test sample solution based on ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, optimizes the sample pretreatment process, chromatographic separation conditions and mass spectrometry detection parameters, and realizes the rapid, sensitive and accurate determination of trehalose and sucrose in monoclonal antibody preparations. The present invention solves the problems that the existing high performance liquid chromatography method can only detect trehalose or sucrose in monoclonal antibodies singly with low analysis efficiency and overcomes the problem of mass spectrometry signal cross-interference, ensuring the accuracy of the quantification of the target substance. In addition, the matrix effect and solvent effect problems are solved by optimizing the pretreatment. The present invention has high sensitivity, high accuracy and good repeatability, and provides an innovative solution for the quality control and stability study 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 cryoprotectants 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 cryoprotectants are widely added to mAb formulations to stabilize the protein structure during lyophilization and storage and prevent its inactivation. Among them, sucrose and trehalose are the most commonly used saccharide cryoprotectants in mAb drugs, which can effectively prevent protein aggregation during freeze-drying and storage by forming a glassy matrix, replacing water molecules, and reducing phase transition 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 cryoprotectants, 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 cryoprotectants in mAb drugs is of great significance for the quality control and stability study 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 cryoprotectants (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 study 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:

[0007] (1) Dilute the test sample with deionized water to obtain a diluted test sample solution;

[0008] (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;

[0009] (3) Dilute the filtrate with acetonitrile to obtain a test sample solution;

[0010] In step (1), the test sample is diluted 50,000 times with deionized water;

[0011] In step (2), the SPE consumable used is Oasis PRiME HLB 96-well µElution Plate, 3 mg;

[0012] In step (3), the volume ratio of the filtrate to acetonitrile is 1:1.

[0013] After preparing the test sample, analyze and detect it using a high-performance liquid chromatography-tandem mass spectrometer.

[0014] The liquid chromatography conditions are as follows: ACQUITY UPLC Glycan BEH Amide Column chromatographic 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.

[0015] 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.

[0016] Table 1. MRM parameter table

[0017] 。

[0018] * is the quantitative ion.

[0019] 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 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.

[0020] The saccharide lyophilization protectants described in the present invention are trehalose and sucrose. In the research and development of monoclonal antibody formulations, common disaccharides may include maltose, lactose, sucrose, and trehalose. Among them, maltose and lactose are often used as ion strength - regulating excipients in products, while trehalose and sucrose are mostly used as lyophilizing agents. Since their molecular structures are similar and their molecular weights are the same, they are prone to generating the same ion channels in mass spectrometry detection, resulting in deviations in quantitative results. Therefore, before the sample enters the mass spectrometer, chromatographic separation technology must be used to ensure that trehalose and sucrose can be fully separated. At the same time, they also need to be separated from their respective isomers to avoid interference with each other during mass spectrometry detection and ensure the accuracy of the detection results.

[0021] The applicant has tried amino columns with silica gel matrices from different manufacturers. The amino group can retain saccharide molecules through hydrophilic interactions, but its silica - amino bonding phase is prone to hydrolysis in an 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 (ACQUITY UPLC Glycan BEH Amide) with a hybrid - particle silica gel matrix 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (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 formulations.

[0024] (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.

[0025] (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, and 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.

[0026] (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

[0027] Figure 1 Chromatogram of a 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;

[0028] Figure 2 Chromatogram of a mixed standard solution of sucrose, maltose, lactose and trehalose in the multi-reaction monitoring (MRM) channels of trehalose and sucrose under the condition of phase A with different concentrations of ammonia water;

[0029] Figure 3 Chromatogram of a 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;

[0030] Figure 4 Chromatogram of a 100 ng / mL mixed standard solution of trehalose and sucrose in the multi-reaction monitoring (MRM) channel of trehalose at different desolvation tube (DL tube) temperatures;

[0031] Figure 5 Chromatogram of a 100 ng / mL mixed standard solution of trehalose and sucrose in the multi-reaction monitoring (MRM) channel of sucrose at different desolvation tube (DL tube) temperatures;

[0032] Figure 6 Chromatogram of a 50 ng / mL mixed standard solution of trehalose and sucrose;

[0033] Figure 7 Chromatogram for actual sample A;

[0034] Figure 8 Chromatogram for actual sample H;

[0035] Figure 9 Chromatogram for actual sample B;

[0036] Figure 10 Overlay diagram for specificity experiment. Detailed implementation manners

[0037] 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.

[0038] 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.

[0039] Example 1

[0040] 1.1. Experimental instruments and equipment

[0041] High-pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

[0042] 1.2. Experimental reagents

[0043] Acetonitrile and ammonia water are of mass spectrometry purity; water is Wahaha purified 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).

[0044] 1.3. Detection conditions

[0045] Liquid chromatography conditions:

[0046] Chromatographic column: ACQUITY UPLC Glycan BEH Amide Column (150 mm×2.1 mm I.D., 1.7μm);

[0047] Mobile phase: A - ammonia water: water (1 - 2:1000, v / v); B - acetonitrile;

[0048] Gradient: 85%B - 65%B from 0 - 10.00 min, 65%B - 50%B from 10.01 - 11.00 min, 85%B from 11.01 - 15.00 min; Column temperature: 50 - 60 °C; Flow rate: 0.25 mL / min; Injection volume: 2.0 μL;

[0049] Mass spectrometry conditions:

[0050] Ion source: Electrospray ionization source, negative ion mode; Mass spectrometer: Triple quadrupole liquid chromatography - mass spectrometry; Desolvation tube temperature: 200 - 300 °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.

[0051] 1.4. Standard curve drawing

[0052] 1) Preparation of standard stock solution: Weigh appropriate amounts of trehalose, sucrose, maltose, and lactose precisely, dissolve them in deionized water to form a stock solution with a concentration of 100 mg / mL each, and then dilute with deionized water to a 1 mg / mL standard stock solution for standby;

[0053] 2) Preparation of standard working solutions: Accurately pipette appropriate amounts of the standard stock solutions of 2 sugars (trehalose, sucrose) respectively, and serially dilute them with acetonitrile - water 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 mixed standard solution of trehalose and sucrose is shown in Figure 6 ;

[0054] 3) Drawing of 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.

[0055] 1.5. Preparation of test sample solution

[0056] (1) Dilute the test sample 50,000 times with deionized water to obtain the diluted test sample solution;

[0057] (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;

[0058] (3) Take 500 μL of the filtrate and dilute it with 500 μL of acetonitrile to obtain the test solution;

[0059] The SPE consumables used in the present invention are Oasis PRiME HLB 96-well µElution Plate, 3 mg.

[0060] 1.6. Determination of the best detection conditions

[0061] In the liquid chromatography conditions, when the column temperature is 50 °C, the baseline separation of trehalose, sucrose and their isomers can be achieved, as Figure 1 shown; when the column temperature is increased to 60 °C, while ensuring the separation effect, the analysis efficiency is significantly improved, and the accurate quantification of trehalose and sucrose in the sample can be completed in only 15 minutes.

[0062] In the present invention, ammonia water is used to adjust the mobile phase to a high pH value condition. During the optimization process of the mobile phase A phase, 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, the A phase was finally determined to be 0.1% ammonia water solution. The mixed standard solution of sucrose, maltose, lactose and trehalose was analyzed under the optimized chromatographic conditions, and the results showed ( Figure 3 ) that in the multiple reaction monitoring (MRM) channels of sucrose and trehalose, maltose and lactose also had signal responses, and their chromatographic peak retention times were highly close and overlapped into one chromatographic peak. The resolution between the two components of sucrose and trehalose and maltose and lactose reached more than 1.5, and the effective separation of trehalose, sucrose and their isomers was successfully achieved. The obtained chromatographic peak baseline was stable, the peak shape was symmetric and sharp, and the signal interference in the mass spectrometry detection was significantly reduced, thus effectively improving the accuracy and reliability of the mass spectrometry quantitative analysis.

[0063] During the optimization process of the mass spectrometry conditions, the temperature of the desolvation tube (DL tube) had a significant impact on the response of the compound. Therefore, the response of the 100 ng / mL mixed standard solution of trehalose and sucrose at different desolvation tube temperatures (200 °C, 250 °C and 300 °C) was investigated, and the chromatogram is shown in Figures 4-5 . The results showed that under different desolvation tube (DL tube) temperature conditions, the response value of trehalose had little difference, while the response value of sucrose increased significantly when the desolvation tube temperature was 250 °C.

[0064] Therefore, the best detection conditions are as follows:

[0065] Liquid chromatography conditions:

[0066] Chromatographic column: ACQUITY UPLC Glycan BEH Amide Column (150 mm×2.1 mm I.D., 1.7 μm);

[0067] Mobile phase: A - ammonia water: water (1:1000, v / v); B - acetonitrile;

[0068] 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;

[0069] Mass spectrometry conditions:

[0070] 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 is nitrogen, flow rate 3.0 L / min; Drying gas is nitrogen, heating gas is air, flow rates are both 10.0 L / min; Mass spectrometry detector detection mode is multiple reaction monitoring (MRM), and the mass spectrometry detection parameters are shown in Table 1.

[0071] Example 2

[0072] 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. Determine 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 RSD. The determination results of the actual samples are shown in Table 2.

[0073] Table 2. Determination results of actual samples (n = 3)

[0074] .

[0075] Note: "N.D." means not detected, and " / " means not calculable.

[0076] Example 3 Specificity experiment

[0077] Since the monoclonal antibody active ingredient in the monoclonal antibody sample to be tested has been purified by SPME and retained in the C18 reverse-phase column, there are three types of specificity verification solutions: blank solution, formulation buffer without trehalose and sucrose, and a mixed standard solution of the reference substance at 10 ng / mL. Among them, ultrapure water is taken as the blank solution; a formulation buffer without trehalose and sucrose is prepared by mixing 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 three specificity verification solutions, indicating that the monoclonal antibody drug itself and other substances in the formulation buffer do not interfere with the detection of trehalose. The results show that this method has high specificity.

[0078] Example 4 Sensitivity Investigation

[0079] The above standard working curve was subjected to linear regression, and the sensitivity of the method was investigated. The correlation coefficients of the obtained calibration curves were all greater than 0.999, and the concentration read-back values of each calibration point were within the range of 95.9% - 104.7% of their theoretical concentrations. The mixed standard solution at the lowest concentration was used for analysis, and the detection limit (S / N = 3) and quantification limit (S / N = 10) were calculated 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 sensitivity within the selected linear range.

[0080] Table 3. Linear Equations, Detection Limits, and Quantification Limits

[0081] 。

[0082] Example 5 Repeatability Investigation

[0083] 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 was between 0.037% and 0.095%, and the RSD of the peak area was between 0.50% and 1.52%. The method has good repeatability.

[0084] Table 4. Repeatability Results of Retention Time and Peak Area (n = 6)

[0085] 。

[0086] Example 6 Recovery Investigation

[0087] The 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 sodium dihydrogen phosphate, 1.1% benzyl alcohol solution) was added with three concentration levels of standard solution: low (series 2), medium (series 4), and high (series 6), and the determination was carried out according to the method established above. Three parallel samples were repeated at each concentration level, and the spike recovery and relative standard deviation (RSD) were calculated. The results are shown in Table 5. The spike recovery of the tested sugar compounds was between 91.3% and 98.5%, and the RSD was between 0.80% and 2.25%.

[0088] Table 5. Spike recovery results (n=3)

[0089] .

[0090] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for simultaneously determining multiple saccharide cryoprotectants in monoclonal antibody drugs, characterized in that, The test solution is detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; The liquid chromatography conditions are as follows: ACQUITY UPLC Glycan BEH Amide chromatographic column, column temperature is 50 - 60 °C, mobile phase A is 0.1% - 0.2% ammonia water solution, mobile phase B is acetonitrile, gradient elution, the concentration range of mobile phase B is 85% - 50%; The mass spectrometry conditions are as follows: electrospray ionization source negative ion mode, desolvation tube temperature is 200 - 300 °C, the detection mode of the mass spectrometry detector is multiple ion selection monitoring; The freeze-drying protectant for saccharides is trehalose and sucrose; The preparation of the test solution includes the following steps: (1) The test sample is diluted with deionized water to obtain a diluted test sample solution; (2) The SPE column is activated with methanol and equilibrated with pure water, then the diluted test sample solution is loaded onto the equilibrated SPE column, rinsed with pure water, and the filtrate is collected; the SPE consumable used is Oasis PRiME HLB 96-well µElution Plate, 3 mg; (3) The filtrate is diluted with acetonitrile to obtain the test solution; The gradient elution program is as follows: 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.

2. A method for simultaneously determining multiple saccharide cryoprotective agents in a monoclonal antibody drug according to claim 1, characterized in that, The specification of the chromatographic column is 150 mm × 2.1 mm, 1.7 μm.

3. A method for simultaneously determining multiple saccharide cryoprotective agents in a monoclonal antibody drug according to claim 1, characterized in that, In the liquid chromatography conditions, the injection volume is 2.0 µL.

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

5. A method for simultaneously determining multiple saccharide cryoprotectants in a monoclonal antibody drug according to claim 1, characterized in that, In step (1), the test sample is diluted 50,000 times with deionized water.

6. A method for simultaneously determining multiple saccharide cryoprotectants in a monoclonal antibody drug according to claim 1, characterized in that, In step (3), the volume ratio of the filtrate to acetonitrile is 1:1.

Images