Method for detecting antibiotics in biological sample

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting antibiotics in a biological sample. The method uses liquid chromatography-mass spectrometry for detection, has good specificity, accuracy, sensitivity, reliability and stability, is low in detection limit and quantitation limit, and can be used for detecting antibiotics (such as G418) in biological samples or biological products (such as cetuximab products) or intermediate products generated in the preparation or purification process of the biological samples or biological products.

Description

Technical Field

[0001] This application belongs to the field of analytical chemistry, and particularly relates to a method for detecting antibiotics (such as G418) in biological samples. Background Art

[0002] According to drug production specifications (such as EMA guidelines) and relevant industry standards, the antibiotic residues in biological products or drugs need to be strictly controlled to below the ppm (parts per million) level to ensure the safety and compliance of drugs.

[0003] G418, also known as geneticin, is an aminoglycoside antibiotic (the structure is shown below). It binds to the 70S and 80S ribosomes of prokaryotic and eukaryotic cells, blocks peptide chain elongation, and thus inhibits protein synthesis. When cells are successfully transfected and integrated with the neomycin resistance gene, they can confer resistance to growth in a selective medium containing G418. Therefore, when preparing monoclonal antibodies such as cetuximab, the gene encoding the antibody is usually inserted into the vector together with the resistance gene, and cells that have been successfully transfected and integrated with the target gene are screened out by G418 to obtain a cell line that stably expresses the antibody.

[0004]

[0005] For biological products such as cetuximab prepared by introducing G418, according to the requirements for similar residual solvents in the ICH guidelines, the permitted daily exposure (PDE) value of G418 in human intravenous injections is approximately 40 - 50 ng / ml, specifically about 46.7 ng / ml.

[0006] However, the detection of G418 residues is difficult because of its complex molecular properties, easy binding to impurities in biological samples, interference with detection sensitivity, and the complex matrix in cell culture media, cell lysates, or purified antibody samples, which may mask the target signal. Most of the detection methods for G418 reported in the literature are high-performance liquid chromatography methods. However, the detection limit of liquid-phase methods is relatively high. For example, the quantitative detection limit of G418 by the ultra-high performance liquid chromatography-evaporative light scattering (UPLC-ELSD) method reported by Zhang Xufan et al. ("Establishment of an ultra-high performance liquid chromatography-evaporative light scattering method for the determination of G418 residues in recombinant proteins", Chinese Journal of Biologicals, January 2020, Vol. 33, No. 1) is 0.01 mg / mL, which is much higher than the safe PDE value of G418 in human intravenous injections and does not meet the requirements of the safety detection limit.

[0007] Therefore, a method capable of effectively detecting trace antibiotics (such as G418) is needed to meet the requirements of supervision and product safety control. Summary of the Invention

[0008] The present application provides a method capable of effectively detecting trace antibiotics (such as G418). This method is simple and convenient, with good specificity, accuracy, sensitivity, reliability, and stability, and has low detection limits and quantification limits.

[0009] On the one hand, the present application provides a method for detecting antibiotics (such as G418) in a sample, including using liquid chromatography - mass spectrometry (LC - MS) for detection.

[0010] In some embodiments, the sample is a biological sample.

[0011] In some embodiments, the sample is a biological product (such as a cetuximab product) or an intermediate product generated during its preparation or purification.

[0012] In some embodiments, the method uses the standard addition method to detect the sample. The inventors have found that for biological products (such as cetuximab products) or intermediate products generated during their preparation or purification, due to the relatively complex matrix, the standard addition method can eliminate the matrix effect and protect the reliability of the detection results.

[0013] In some embodiments, since the sample matrix is relatively complex, containing inorganic salts, proteins, etc., the sample is ultrafiltered before detection. The inventors have found that the ultrafiltration operation can remove macromolecular proteins, thereby effectively reducing the contamination of the ion source by protein - based matrix and the influence on the ionization of G418.

[0014] In some embodiments, the cut - off molecular weight of the ultrafiltration operation is 3 - 100 KD, such as 5 - 20 KD, such as about 10 KD.

[0015] In some embodiments, an ultrafiltration centrifuge tube is used for the ultrafiltration operation.

[0016] In some embodiments, the chromatographic column in the liquid chromatography is a hydrophilic chromatographic column.

[0017] In some embodiments, the chromatographic column is selected from silica gel columns, diol - based columns, amino columns, cyano columns, and amide - based columns. In some embodiments, the chromatographic column is an amide - based column.

[0018] In some embodiments, the inner diameter of the chromatographic column in the liquid chromatography is 1 - 8 mm, such as 1 - 5 mm, such as about 2.1 mm.

[0019] In some embodiments, the length of the chromatographic column in the liquid chromatography is 50 - 300 mm, such as about 150 mm.

[0020] In some embodiments, the particle size of the packing material of the chromatographic column in the liquid chromatography is 0.5 - 8 μm, such as about 1.7 μm.

[0021] In some embodiments, the chromatographic column in the liquid chromatography is a Waters ACQUITY UPLC GlycanBEH Amide chromatographic column, preferably having the following specifications: Waters ACQUITY UPLC Glycan BEH Amide Column130Å 2.1mm×150mm, 1.7μm.

[0022] In some embodiments, the mobile phase of the liquid chromatography is a mixture of mobile phase A and mobile phase B.

[0023] In some embodiments, mobile phase A is an aqueous solution containing a buffer salt. In some embodiments, the buffer salt is selected from formates, such as ammonium formate; acetates, such as ammonium acetate; phosphates, such as sodium phosphate or potassium phosphate, etc.

[0024] In some embodiments, mobile phase A further contains acids. In some embodiments, the acids are trifluoroacetic acid, formic acid, acetic acid or phosphoric acid.

[0025] In some embodiments, mobile phase A is an aqueous solution containing ammonium formate and formic acid.

[0026] In some embodiments, the concentration of ammonium formate contained in mobile phase A is 0.001 - 0.3 mol / L, for example, about 0.01 - 0.1 mol / L, for example, about 0.03 mol / L. The inventors have found that when the concentration of the buffer solution in the mobile phase is relatively high, the detection peak shape is better.

[0027] In some embodiments, the concentration of formic acid contained in mobile phase A is 0.01 - 0.5% (v / v), preferably 0.1 - 0.3% (v / v), for example, about 0.2% (v / v).

[0028] In some embodiments, mobile phase A is a solution of about 0.03 mol / L ammonium formate, which contains about 0.2% (v / v) formic acid.

[0029] In some embodiments, mobile phase B is an organic solvent or a mixed system of an organic solvent and water. In some embodiments, the organic solvent is selected from one or more of acetonitrile, methanol and tetrahydrofuran, preferably acetonitrile. In some embodiments, mobile phase B is an acetonitrile - water system. In some embodiments, the acetonitrile - water system contains 60 - 100% (v / v) acetonitrile, for example, 70 - 90% (v / v), and further for example, about 80% (v / v).

[0030] In some embodiments, the mobile phase B contains a buffer salt. In some embodiments, the buffer salt is selected from formates such as ammonium formate; acetates such as ammonium acetate; phosphates such as sodium phosphate or potassium phosphate, etc.

[0031] In some embodiments, the mobile phase B further contains acids. In some embodiments, the acids are trifluoroacetic acid, formic acid, acetic acid or phosphoric acid.

[0032] In some embodiments, the mobile phase B is an acetonitrile - water system containing a buffer salt and acids. In some embodiments, the mobile phase B is an acetonitrile - water system containing ammonium formate and formic acid.

[0033] In some embodiments, the concentration of ammonium formate contained in the mobile phase B is 0.001 - 0.3 mol / L, for example 0.01 - 0.1 mol / L, for example about 0.03 mol / L.

[0034] In some embodiments, the concentration of formic acid contained in the mobile phase B is 0.01 - 0.5% (v / v), preferably 0.1 - 0.3% (v / v), for example about 0.2% (v / v).

[0035] In some embodiments, the mobile phase B is an 80% acetonitrile solution of about 0.03 mol / L ammonium formate, which contains about 0.2% (v / v) formic acid.

[0036] In some embodiments, the flow rate is 0.2 - 1.5 ml / min, for example about 0.6 ml / min.

[0037] In some embodiments, the column temperature is 20 - 60 °C, preferably 40 - 55 °C, for example about 50 °C.

[0038] In some embodiments, the liquid chromatography method uses gradient elution.

[0039] In some embodiments, the gradient elution program is as follows: the initial gradient is that the volume percentage of the mobile phase B in the mobile phase is 80% - 100%, the second gradient is that the volume percentage of the mobile phase B in the mobile phase changes from 80% - 100% to 60 - 80%, the third gradient is that the volume percentage of the mobile phase B in the mobile phase is 60 - 80%, the fourth gradient is that the volume percentage of the mobile phase B in the mobile phase changes from 60 - 80% to 80% - 100%, and the final gradient is that the volume percentage of the mobile phase B in the mobile phase is 80% - 100%; the elution time of the initial gradient is 1 - 3 min, the elution time of the second gradient is 0.5 - 2 min, the elution time of the third gradient is 2 - 6 min, the elution time of the fourth gradient is 0 - 1 min, and the elution time of the final gradient is 2 - 5 min.

[0040] In some embodiments, the gradient elution procedure is as follows:

[0041]

[0042] In some embodiments, the mass spectrometry uses an electrospray ionization source (ESI).

[0043] In some embodiments, the mass spectrometry performs mass scanning in the positive ion multiple reaction monitoring mode MRM.

[0044] In some embodiments, the declustering voltage in the mass spectrometry is 100 - 160 V, such as 120 - 140 V, such as about 130 V.

[0045] In some embodiments, the drying gas flow rate in the mass spectrometry is 5 - 15 L / min, such as 8 - 12 L / min, such as about 9 - 11 L / min, such as about 10 L / min.

[0046] In some embodiments, the drying gas temperature in the mass spectrometry is 200 - 500 °C, such as 300 - 400 °C, such as about 330 - 350 °C, such as about 350 °C.

[0047] In some embodiments, the nebulizing gas pressure in the mass spectrometry is 20 - 50 psi, such as 40 - 45 psi, such as about 35 psi.

[0048] In some embodiments, the sheath gas temperature in the mass spectrometry is 200 - 500 °C, such as 300 - 400 °C, such as 330 - 370 °C, such as about 350 °C.

[0049] In some embodiments, the sheath gas flow rate in the mass spectrometry is 6 - 18 L / min, such as 10 - 15 L / min, such as 11 - 12 L / min, such as about 12 L / min.

[0050] In some embodiments, the capillary voltage in the mass spectrometry is +1500 V to +3500 V, such as +2000 V to +3000 V, such as about +2500 V.

[0051] In some embodiments, the nozzle voltage in the mass spectrometry is +300 V to +700 V, such as +400 V to +600 V, such as about +500 V.

[0052] In some embodiments, the detector gain voltage in the mass spectrometry is 150 - 250 V, such as about 200 V.

[0053] In some embodiments, the collision cell acceleration voltage in the mass spectrometry is 2 - 6 V, such as about 4 V.

[0054] In some embodiments, the dwell time in the mass spectrometry is 50 - 150 ms, such as 80 - 120 ms, such as about 100 ms.

[0055] In some embodiments, the precursor ion for the G418 mass spectrometry detection is 497.3 (m / z).

[0056] In some embodiments, the product ions for the G418 mass spectrometry detection are selected from 380.3, 338.3, 205.1, 163.1 (m / z).

[0057] In some embodiments, the qualitative or quantitative ion pairs of G418 are selected from one or more of the following: 497.3 → 380.3; 497.3 → 338.3; 497.3 → 205.1; 497.3 → 163.1.

[0058] In some embodiments, the qualitative ion pairs of G418 are selected from one or more of the following: 497.3 → 380.3; 497.3 → 338.3; 497.3 → 205.1; and the quantitative ion pair of G418 is 497.3 → 163.1.

[0059] In some embodiments, the collision energy (CE) in the mass spectrometry is 5 - 40 eV.

[0060] In some embodiments, the collision energy (CE) for the ion pair 497.3 → 380.3 in the mass spectrometry is 10 - 20 eV, such as about 16 eV.

[0061] In some embodiments, the collision energy (CE) for the ion pair 497.3 → 338.3 in the mass spectrometry is 10 - 20 eV, such as about 16 eV.

[0062] In some embodiments, the collision energy (CE) for the ion pair 497.3 → 205.1 in the mass spectrometry is 20 - 30 eV, such as about 24 eV.

[0063] In some embodiments, the collision energy (CE) for the ion pair 497.3 → 163.1 in the mass spectrometry is 20 - 40 eV, such as about 28 eV.

[0064] The beneficial effects of the present application include: The method for detecting G418 in the present application is simple and convenient, with good specificity, accuracy, sensitivity, reliability and stability, and has low detection limit and quantification limit, and can effectively detect the content of G418 in biological products and intermediate products generated during their preparation and / or purification processes, and the detection limit can even be as low as ng / mL.

[0065] Those skilled in the art can understand that the method of the present application can also be used to detect other antibiotics in the sample other than G418, such as neomycin, etc.

[0066] All technical features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive technical features and / or steps, can be combined in any manner.

[0067] Unless otherwise specified, the numerical values in the present application are all modified by the term "about". The term "about" means within ±20% of the stated numerical value, preferably within ±10%, more preferably within ±5%, and even more preferably within ±2%. Description of the Drawings

[0068] Figure 1 It is the detection result of Example 1.

[0069] Figure 2 It is the detection result of Comparative Example 1.

[0070] Figure 3 It is the detection result of Comparative Example 2.

[0071] Figure 4 Exemplarily shows the detection chromatogram of the blank test sample solution in Example 3.

[0072] Figure 5 Exemplarily shows the detection chromatogram of the reference solution in Example 3.

[0073] Figure 6 Exemplarily shows the detection chromatogram of the sample in Example 9. Detailed Description of the Invention

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the present specification (including definitions) shall prevail.

[0075] Throughout the specification and claims, the word "comprising" will be understood to include the stated items, but not to exclude any other alternatives. Any examples following "for example" or "such as" are not meant to be exhaustive or limiting.

[0076] The present application includes the following embodiments: Embodiment 1. A method for detecting G418 in a sample, characterized in that the method includes using liquid chromatography - mass spectrometry (LC - MS) for detection, and the chromatographic column in the liquid chromatography is a hydrophilic chromatographic column.

[0077] Embodiment 2. The method according to Embodiment 1, wherein the chromatographic column is selected from a silica gel column, a diol column, an amino column, a cyano column, and an amide column, preferably an amide column.

[0078] Embodiment 3. The method according to Embodiment 1 or 2, wherein the mobile phase used in the liquid chromatography is a mixture of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing a buffer salt and an acid, and mobile phase B is an organic solvent optionally containing a buffer salt and an acid or a mixed system of an organic solvent and water; Preferably, the buffer salt is selected from: formate, such as ammonium formate; acetate, such as ammonium acetate; phosphate, such as sodium phosphate or potassium phosphate; The acid is trifluoroacetic acid, formic acid, acetic acid, or phosphoric acid; The organic solvent is selected from one or more of acetonitrile, methanol, and tetrahydrofuran, preferably acetonitrile.

[0079] Embodiment 4. The method according to any one of Embodiments 1-3, wherein mobile phase A is an aqueous solution containing ammonium formate and formic acid; mobile phase B is an acetonitrile-water system containing ammonium formate and formic acid; Preferably, the concentration of ammonium formate in mobile phase A is 0.001 to 0.3 mol / L, such as 0.01 to 0.1 mol / L, such as about 0.03 mol / L, and the concentration of formic acid in mobile phase A is 0.01 to 0.5% (v / v), preferably 0.1 to 0.3% (v / v), such as about 0.2% (v / v); The concentration of ammonium formate in mobile phase B is 0.001 to 0.3 mol / L, such as 0.01 to 0.1 mol / L, such as about 0.03 mol / L; the concentration of formic acid in mobile phase B is 0.01 to 0.5% (v / v), preferably 0.1 to 0.3% (v / v), such as about 0.2% (v / v); The acetonitrile-water system contains 60 to 100% (v / v) of acetonitrile, such as 70 to 90% (v / v), and further such as about 80% (v / v).

[0080] Embodiment 5. The method according to any one of Embodiments 1-4, wherein the liquid chromatography method uses gradient elution, preferably using the following gradient elution program: the initial gradient is that the volume percentage of mobile phase B in the mobile phase is 80% - 100%, the second gradient is that the volume percentage of mobile phase B in the mobile phase changes from 80% - 100% to 60 - 80%, the third gradient is that the volume percentage of mobile phase B in the mobile phase is 60 - 80%, the fourth gradient is that the volume percentage of mobile phase B in the mobile phase changes from 60 - 80% to 80% - 100%, and the final gradient is that the volume percentage of mobile phase B in the mobile phase is 80% - 100%; the elution time of the initial gradient is 1 - 3 min, the elution time of the second gradient is 0.5 - 2 min, the elution time of the third gradient is 2 - 6 min, the elution time of the fourth gradient is 0 - 1 min, and the elution time of the final gradient is 2 - 5 min; More preferably, the following gradient elution program is used:

[0081] Embodiment 6. The method according to any one of Embodiments 1-5, wherein the mass spectrometry uses an electrospray ionization source ESI and performs mass scanning in the positive ion multiple reaction monitoring mode MRM; Preferably, it includes one or more of the following features: (1) The declustering voltage is 100 - 160 V, such as 120 - 140 V, such as about 130 V; (2) The precursor ion for G418 mass spectrometry detection is 497.3 (m / z); (3) The product ions for G418 mass spectrometry detection are selected from 380.3, 338.3, 205.1, 163.1 (m / z); preferably, the qualitative ion pairs of G418 are selected from one or more of the following: 497.3→380.3; 497.3→338.3; 497.3→205.1; the quantitative ion pair of G418 is 497.3→163.1; (4) The collision energy (CE) is 5 - 40 eV. Preferably, for the ion pair 497.3→380.3, the collision energy (CE) is 10 - 20 eV, such as about 16 eV; for the ion pair 497.3→338.3, the collision energy (CE) is 10 - 20 eV, such as about 16 eV; for the ion pair 497.3→205.1, the collision energy (CE) is 20 - 30 eV, such as about 24 eV; for the ion pair 497.3→163.1, the collision energy (CE) is 20 - 40 eV, such as about 28 eV.

[0082] Embodiment 7. The method according to any one of Embodiments 1-6, wherein the method includes one or more of the following features: (1) The inner diameter of the chromatographic column in the liquid chromatography is 1 to 8 mm, such as 1 to 5 mm, such as about 2.1 mm; (2) The length of the chromatographic column in the liquid chromatography is 50 to 300 mm, such as about 150 mm; (3) The particle size of the packing material of the chromatographic column in the liquid chromatography is 0.5 to 8 μm, such as about 1.7 μm; (4) The chromatographic column in the liquid chromatography is a Waters ACQUITY UPLC Glycan BEH Amide chromatographic column, preferably having the following specifications: Waters ACQUITY UPLC Glycan BEH Amide Column 130Å 2.1mm×150mm, 1.7μm; (5) The drying gas flow rate is 5 to 15 L / min, such as 8 to 12 L / min, such as about 9 to 11 L / min, such as about 10 L / min; (6) The drying gas temperature is 200 to 500 °C, such as 300 to 400 °C, such as about 330 to 350 °C, such as about 350 °C; (7) The nebulizing gas pressure is 20 to 50 psi, such as 40 to 45 psi, such as about 35 psi; (8) The sheath gas temperature is 200 to 500 °C, such as 300 to 400 °C, 330 to 370 °C, such as about 350 °C; (9) The sheath gas flow rate is 6 to 18 L / min, such as 10 to 15 L / min, such as 11 to 12 L / min, such as about 12 L / min; (10) The capillary voltage is +1500 V to +3500 V, such as +2000 V to +3000 V, such as about +2500 V; (11) The nozzle voltage is +300 V to +700 V, such as +400 V to +600 V, such as about +500 V; (12) The detector gain voltage is 150 to 250 V, such as about 200 V; (13) The collision cell acceleration voltage is 2 to 6 V, such as about 4 V; (14) The dwell time is 50 to 150 ms, such as 80 to 120 ms, such as about 100 ms.

[0083] Embodiment 8. The method according to any one of Embodiments 1-7, wherein the sample is a biological product (such as a cetuximab product) or an intermediate product generated during its preparation or purification.

[0084] Embodiment 9. The method according to any one of Embodiments 1-8, wherein the method uses the standard addition method to detect the sample.

[0085] Embodiment 10. The method according to any one of Embodiments 1-9, wherein the sample is ultrafiltered before detection, preferably the cut-off molecular weight of the ultrafiltration operation is 3-100 KD, such as 5-20 KD, such as about 10 KD.

[0086] Embodiment 11. Use of the method according to any one of Embodiments 1-10 for detecting G418 in biological products (such as cetuximab products) or intermediate products generated during their preparation or purification.

[0087] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention. For those specific technologies or conditions not specified in the embodiments, the technologies or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase. Unless otherwise specified, the percentages used in the embodiments are volume percentages.

[0088] Instruments and materials used in the examples:

[0089]

[0090] Note: The AEX test sample stock solution refers to the solution system obtained after the anion exchange (AEX) chromatography step during the preparation of biological products such as cetuximab; the CEX test sample stock solution refers to the solution system obtained after the cation exchange (CEX) chromatography step during the preparation of biological products such as cetuximab; the UF test sample stock solution refers to the solution system obtained after the ultrafiltration (UF) step during the preparation of biological products such as cetuximab; the AC test sample stock solution refers to the solution system obtained after the activated carbon treatment (AC) during the preparation of biological products such as cetuximab.

[0091] Example 1 Mass spectrometry conditions

[0092] 1.1 Establishment of precursor ions

[0093] Instrument: Agilent1290 - Agilent 6470

[0094] Chromatographic conditions: Chromatographic column: Agilent RRHD SB-C18 2.1×100mm, 1.8μm; Mobile phase: 0.1% formic acid solution - acetonitrile (40:60), Injection volume: 1μl; Flow rate: 0.3ml / min; Column temperature: 25°C; Run for 5 min.

[0095] Mass spectrometry conditions: Ion source: AJS-ESI; Monitoring mode: MS2 SCAN; Dry gas flow rate: 10 L / min; Dry gas temperature: 350 °C; Nebulizer gas pressure: 35 psi; Sheath gas temperature: 350 °C; Sheath gas flow rate: 12 L / min; Declustering voltage: 135 V; Capillary voltage: +2500 V; Starting mass (m / z): 300; Ending mass (m / z): 500.

[0096] Reference stock solution: Weigh 1.556 mg of G418 and add water to 25 ml.

[0097] Inject the reference stock solution for analysis to obtain the precursor ion (m / z) of G418 as 497.3.

[0098] 1.2 Determination of declustering voltage value

[0099] Monitoring mode: MS2 SIM; Precursor ion: 497.3; Declustering voltage: 10 V - 200 V, step: 10 V. Other conditions are the same as 1.1.

[0100] Inject the reference stock solution for analysis to obtain the optimal declustering voltage of G418 as 130 V.

[0101] 1.3 Determination of product ions

[0102] Monitoring mode: Product Ion; MS2 scan range: 30 - 500; CE: 5 eV, 15 eV, 25 eV, 35 eV; Declustering voltage: 130 V; Other conditions are the same as 1.1.

[0103] Inject the reference stock solution for analysis to obtain the product ions (m / z) of G418 as 380.3, 338.3, 205.1, 163.1.

[0104] 1.4 Determination of collision energy (CE) value

[0105] Monitoring mode: Multiple reaction monitoring (MRM); CE optimization range: 2 - 60 eV, step: 2 eV; Other conditions are the same as 1.1.

[0106] Inject the reference stock solution for analysis to obtain the optimal CE value, as shown in Table 1 specifically.

[0107] Table 1 Mass spectrometry parameters of G418

[0108]

[0109] Example 2

[0110] Prepare the reference solution: Weigh an appropriate amount of G418 and dilute it with the basal medium solution. Then analyze this reference solution using the following conditions.

[0111] Chromatographic conditions: Chromatographic column: Waters ACQUITY UPLC Glycan BEH Amide Column 130Å 2.1 mm × 150 mm, 1.7 μm; Mobile phase A: 0.03 mol / L ammonium formate solution (containing 0.2% formic acid); Mobile phase B: 80% acetonitrile solution of 0.03 mol / L ammonium formate (containing 0.2% formic acid); Column temperature: 50 °C; Injection volume: 5 μl; Flow rate: 0.6 ml / min; The gradient program is shown in Table 2 below:

[0112] Table 2 Elution program

[0113]

[0114] Mass spectrometry conditions: Ion source parameters: Ion source: AJS-ESI; Scanning mode: MRM; Dry gas flow rate: 10 L / min; Dry gas temperature: 350 °C; Nebulizing gas pressure: 35 psi; Sheath gas temperature: 350 °C; Sheath gas flow rate: 12 L / min; Capillary voltage: +2500 V; Nozzle voltage: +500 V; Detector gain voltage: 200 V; Collision cell acceleration voltage: 4 V; Dwell time: 100 ms; The collision energy (CE), etc. are shown in Table 3 below.

[0115] Table 3 Mass spectrometry parameters

[0116]

[0117] See the detection results in Figure 1 . The results show that under the above conditions, G418 has good retention and response.

[0118] Example 3

[0119] Liquid phase conditions:

[0120] Chromatographic column: Waters ACQUITY UPLC Glycan BEH Amide Column 130Å 2.1 mm × 150 mm, 1.7 μm; Mobile phase A: 0.03 mol / L ammonium formate solution (containing 0.2% formic acid); Mobile phase B: 80% acetonitrile solution of 0.03 mol / L ammonium formate (containing 0.2% formic acid); Column temperature: 50 °C; Injection volume: 10 μl (AC solution system), 5 μl (AEX, CEX, UF solution system); Injector temperature: 5 °C; Flow rate: 0.6 ml / min; The gradient program is shown in Table 4.

[0121] Table 4 Elution program

[0122]

[0123] Mass spectrometry conditions: Ion source: AJS-ESI; Scan type: MRM; Dry gas flow rate: 10 L / min; Dry gas temperature: 350 °C; Nebulizer gas pressure: 35 psi; Sheath gas temperature: 350 °C; Sheath gas flow rate: 12 L / min; Capillary voltage: +2500 V; Nozzle voltage: +500 V; Detector gain voltage: 200 V; Collision cell acceleration voltage: 4 V; Dwell time: 100 ms; Collision energy (CE), etc. See Table 5 below.

[0124] Table 5 Mass spectrometry parameters

[0125]

[0126] Sample preparation:

[0127] Test stock solutions: Include AEX test stock solution, CEX test stock solution, UF test stock solution, and AC test stock solution.

[0128] Reference stock solution: Weigh about 0.27 mg of G418 → 10 ml; 0.5 ml → 10 ml; 1 ml → 20 ml (basic culture medium solution, about 46.7 ng / ml).

[0129] Reference solution: Take 0.8 ml of the reference stock solution + 0.8 ml of the test stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15000 rpm for 10 min, and take the lower layer of the supernatant.

[0130] Blank test solution: Take 0.8 ml of the test stock solution without G418 → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15000 rpm for 10 min, and take the lower layer of the supernatant.

[0131] Test solution: Take 0.8 ml of the test stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15000 rpm for 10 min, and take the lower layer of the supernatant.

[0132] 100% recovery solution: Take 0.8 ml of the test stock solution + 0.8 ml of the reference stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15000 rpm for 10 min, and take the lower layer of the supernatant.

[0133] Injection analysis and results:

[0134] Inject the blank test solution, reference solution, test solution, and 100% recovery solution for analysis. If there is a peak in the test solution chromatogram with the same retention time as G418 in the reference solution chromatogram, calculate it by the external standard method using the peak area.

[0135] Calculation formula:

[0136]

[0137] In the formula: W S is the weighed amount of the reference substance (mg); V T is the sampling volume of the test sample (ml)

[0138] D S and D T are the dilution factors of the reference substance and the test sample respectively;

[0139] A S is the peak area after subtracting the test sample peak area from the reference substance peak area;

[0140] A T is the peak area of the test sample; p is the content of the reference substance.

[0141] The results show that there is no interference in the blank test solutions at the peak position of G418. Among them, Figure 4 exemplarily shows the detection chromatogram of the blank AEX test solution, Figure 5 exemplarily shows the detection chromatogram of the reference solution; and the sample addition recoveries of each system are all between 85% and 115%. Among them, the recovery rate of the AEX solution system is 99.18%, the recovery rate of the CEX solution system is 97.35%, the recovery rate of the UF system is 95.98%, and the recovery rate of the AC system is 108.32%.

[0142] Example 4 Linearity and Range

[0143] Sample preparation:

[0144] Reference stock solution: Weigh about 0.27 mg of G418 → 10 ml; 0.5 ml → 10 ml (basic medium solution, about 934 ng / ml).

[0145] Linear solution: Take an appropriate amount of the reference stock solution, dilute it with the basic medium solution to prepare a series of linear stock solutions. Take 0.8 ml of each series of linear stock solutions and place them in a 2-ml volumetric flask. Add 0.8 ml of each test sample stock solution to each flask, then dilute to the mark with the basic medium solution. Mix well and take appropriate amounts, place them in ultrafiltration centrifuge tubes (10KD) respectively, centrifuge at 15000 rpm for 10 min, and take the lower-layer clear liquid.

[0146] Using the LC-MS detection conditions in Example 3, samples were injected for analysis from low concentration to high concentration. Under each solution system, the regression curve and correlation coefficient were calculated based on the concentration and the corresponding G418 peak area. The results are shown in Table 6 below.

[0147] Table 6 Linearity and Range

[0148]

[0149] The results showed that under each solution system, the correlation coefficient r of G418 was greater than 0.990, and the intercept deviation was small, all less than 10%.

[0150] Example 5 Detection Limit and Quantification Limit

[0151] According to the test results of Example 4, the detection limit concentration was determined with a signal-to-noise ratio ≥ 3, and the quantification limit concentration was determined with a signal-to-noise ratio ≥ 10. The results are shown in Table 7 below.

[0152] Table 7 Investigation Results of the Detection Limit and Quantification Limit of the G418 Detection Method under Each Solution System

[0153]

[0154] Note: The limit is 46.7 ng / ml.

[0155] The results showed that under each solution system, the quantification limit of G418 was less than 30% of the limit concentration, and the detection limit was less than 20% of the limit concentration.

[0156] Example 6 Accuracy and Precision

[0157] Reference stock solution: Appropriate amount of G418 was taken and diluted to about 46.7 ng / ml with the basic culture medium solution.

[0158] Reference solution: Take 0.8 ml of the reference stock solution + 0.8 ml of the test sample stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15,000 rpm for 10 min, and take the lower layer of the supernatant.

[0159] Blank test sample solution: Take 0.8 ml of the test sample stock solution without G418 → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15,000 rpm for 10 min, and take the lower layer of the supernatant.

[0160] Test sample solution: Take 0.8 ml of the test sample stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10KD), centrifuge at 15,000 rpm for 10 min, and take the lower layer of the supernatant.

[0161] 50% recovery solution: Take 0.8 ml of the test sample stock solution + 0.4 ml of the control stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0162] 100% recovery solution: Take 0.8 ml of the test sample stock solution + 0.8 ml of the control stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0163] 150% recovery solution: Take 0.8 ml of the test sample stock solution + 1.2 ml of the control stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0164] Take each blank test sample solution, control solution, test sample solution and each recovery solution, inject them for analysis, and the results are shown in Table 8.

[0165] Table 8 Investigation results of the accuracy and precision of the G418 detection method under each solution system (the base is not detected)

[0166]

[0167] The results show that under each solution system, when G418 with a concentration level of 50% - 150% of the limit concentration is added to the test sample solution, the recovery rates are all between 85% and 115%, and the RSD (n = 9) are all < 15%.

[0168] Example 7 Solution Stability

[0169] Sample preparation:

[0170] Control stock solution: Weigh about 0.27 mg of G418 → 10 ml; 0.5 ml → 10 ml; 1 ml → 20 ml (basic culture medium solution, about 46.7 ng / ml).

[0171] Control solution: Take 0.8 ml of the control stock solution + 0.8 ml of the test sample stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0172] Test sample solution: Take 0.8 ml of the test sample stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0173] 100% recovery solution: Take 0.8 ml of the test sample stock solution + 0.4 ml of the reference stock solution → 2 ml (basic culture medium solution), mix well, take an appropriate amount, place it in an ultrafiltration centrifugal tube (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0174] Inject and analyze the reference solution and the recovery solution under each solution system at 5 °C for different times. The results are shown in Table 9.

[0175] Table 9 Results of the investigation on the solution stability of the G418 detection method under each solution system

[0176]

[0177] Result: Within 8 - 11 h of investigation at 5 °C, there was no significant change in the peak area of G418 in the reference solution and the test sample solution compared with that at 0 h (the peak area change rate was < 20%), indicating that the reference solution and the test sample solution were stable within the corresponding investigation time at 5 °C.

[0178] Example 8 Method durability

[0179] Prepare the reference solution, test sample solution, and recovery solution for the investigation of durability under each system in the same way as in Example 7.

[0180] Under the basic LC-MS conditions of Example 3, fine-tune the liquid phase and mass spectrometry conditions respectively, and inject and analyze. The results are shown in Table 10.

[0181] Table 10 Results of the investigation on the durability of the G418 detection method under each solution system

[0182]

[0183] Result: By fine-tuning the relevant chromatographic and mass spectrometry parameters, the blank test sample solution under each solution system did not interfere with the determination of G418, and there was no significant difference in the recovery rate (RSD < 15%, n = 5), indicating good method durability.

[0184] Example 9

[0185] Use the LC-MS detection conditions of Example 3 to detect a sample, and the detected amount of G418 in the sample is 3.6 ng / ml. Figure 6 Exemplarily show the detection spectrum of the sample.

[0186] Comparative Example 1

[0187] Inject and analyze the reference stock solution of Example 1 under conditions similar to those of Example 2, with the difference that the following chromatographic conditions are used.

[0188] Chromatographic conditions: Chromatographic column: Agilent RRHD SB-C18 2.1×100 mm, 1.8 μm; Mobile phase A: 0.1% formic acid solution; Mobile phase B: acetonitrile; Column temperature: 30 °C; Injection volume: 2 μl; Mobile phase A - Mobile phase B (40:60), Flow rate: 0.3 ml / min, Run for 5 min.

[0189] See the detection results in Figure 2 . The results showed that G418 had almost no retention. In samples with a more complex matrix, due to the presence of non-retained matrix such as inorganic salts, using this chromatographic condition was likely to produce matrix effects and contaminate the ion source at the same time.

[0190] Comparative Example 2

[0191] The reference substance stock solution of Example 1 was injected and analyzed under conditions similar to those of Comparative Example 1, except that the following gradient elution conditions were used.

[0192]

[0193] See the detection results in Figure 3 . The results showed that G418 had almost no retention.

[0194] Comparative Example 3

[0195] The test sample solution was detected using the LC-MS detection conditions of Example 3, except that after diluting the original test sample solution, it was directly injected for analysis without performing an ultrafiltration operation.

[0196] It was found during the detection process that the pressure of the chromatographic column increased significantly, indicating that the chromatographic column was blocked and the detection could not be completed.

[0197] Comparative Example 4

[0198] The test sample solution was detected using the LC-MS detection conditions of Example 3, except that the standard addition method was not used and the external standard method was adopted. It was found that during the detection using the external standard method, due to the complex matrix of the test sample, there was a significant inhibitory effect on the response of G418, and the spiked recovery rate of G418 was significantly low in some systems. The results are shown in Table 11, while using the standard addition method as shown in Example 3 can effectively avoid the interference caused by the matrix.

[0199] Table 11 Results of the investigation of the spiked recovery of G418 in each solution system using the external standard method (the base was not detected in all cases)

[0200]

[0201] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. All the content disclosed in the specification, including the abstract, and all the methods and steps disclosed can be combined arbitrarily, unless these features and / or steps are mutually exclusive combinations. Each technical feature disclosed in the specification, including the abstract, unless otherwise stated, can be replaced by technical features that achieve the same, equivalent or similar purposes. Therefore, unless otherwise stated, each technical feature disclosed in the present invention is only an example of equivalent or similar technical features in the general series. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting G418 in a sample, characterized in that: The method comprises the step of using liquid chromatography-mass spectrometry (LC-MS) for detection, wherein the chromatographic column in the liquid chromatography is a hydrophilic chromatographic column.

2. The method of claim 1, wherein the sample is a biological product or an intermediate product produced during its preparation or purification.

3. The method according to claim 1, wherein the sample is a cetuximab product or an intermediate product produced during its preparation or purification.

4. The method according to any one of claims 1 to 3, wherein the chromatographic column is selected from the group consisting of a silica gel column, a diol column, an amino column, a cyano column and an amide column.

5. The method of claim 4, wherein the chromatographic column is an amide column.

6. The method according to any one of claims 1 to 3, wherein the mobile phase used in the liquid chromatography is a mixture of mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing a buffer salt and an acid, and the mobile phase B is an organic solvent or a mixed system of an organic solvent and water optionally containing a buffer salt and an acid; The buffer salt is selected from the group consisting of formate, acetate and phosphate; The acid is trifluoroacetic acid, formic acid, acetic acid or phosphoric acid; The organic solvent is selected from one or more of acetonitrile, methanol and tetrahydrofuran.

7. The method according to any one of claims 1 to 3, wherein mobile phase A is an aqueous solution containing ammonium formate and formic acid; and mobile phase B is an acetonitrile-water system containing ammonium formate and formic acid.

8. The method according to claim 7, wherein the concentration of ammonium formate contained in the mobile phase A is 0.001-0.3 mol / L, and the concentration of formic acid contained in the mobile phase A is 0.01-0.5% (v / v); The concentration of ammonium formate contained in mobile phase B is 0.001~0.3 mol / L; the concentration of formic acid contained in mobile phase B is 0.01~0.5% (v / v); The acetonitrile-water system contains 60-100% (v / v) acetonitrile.

9. The method according to claim 8, wherein the concentration of ammonium formate contained in the mobile phase A is 0.01-0.1 mol / L, and the concentration of formic acid contained in the mobile phase A is 0.1-0.3% (v / v); The concentration of ammonium formate contained in mobile phase B is 0.01~0.1 mol / L; the concentration of formic acid contained in mobile phase B is 0.1~0.3% (v / v); The acetonitrile-water system contains 70-90% (v / v) acetonitrile.

10. The method of claim 8, wherein the concentration of ammonium formate contained in the mobile phase A is 0.03 mol / L, and the concentration of formic acid contained in the mobile phase A is 0.2% (v / v); The concentration of ammonium formate contained in mobile phase B was 0.03 mol / L; the concentration of formic acid contained in mobile phase B was 0.2% (v / v); The acetonitrile-water system contains 80% (v / v) acetonitrile.

11. The method according to any one of claims 1 to 3, wherein the liquid chromatography method adopts gradient elution, and the following gradient elution program is adopted: the initial gradient is that the volume percentage of mobile phase B in the mobile phase is 80% to 100%, the second gradient is that the volume percentage of mobile phase B in the mobile phase changes from 80% to 100% to 60-80%, the third gradient is that the volume percentage of mobile phase B in the mobile phase is 60-80%, the fourth gradient is that the volume percentage of mobile phase B in the mobile phase changes from 60-80% to 80% to 100%, and the final gradient is that the volume percentage of mobile phase B in the mobile phase is 80% to 100%; the elution time of the initial gradient is 1 to 3 min, the elution time of the second gradient is 0.5 to 2 min, the elution time of the third gradient is 2 to 6 min, the elution time of the fourth gradient is 0 to 1 min, and the elution time of the final gradient is 2 to 5 min.

12. The method of claim 11, wherein the following gradient elution program is used: 。 13. The method according to any one of claims 1 to 3, wherein the mass spectrometer uses an electrospray ion source (ESI) and a positive ion multiple reaction monitoring (MRM) mode is used for mass scanning.

14. The method of claim 13, wherein the method comprises one or more of the following features: (1) Declustering voltage is 100~160V; (2) The precursor ion detected by G418 mass spectrometry is 497.3 (m / z); (3) Product ions detected by G418 mass spectrometry are selected from 380.3, 338.3, 205.1, and 163.1 (m / z); (4) The collision energy is 5~40eV.

15. The method of claim 14, wherein the method comprises one or more of the following features: (1) Declustering voltage is 120~140V; (2) The qualitative ion pair of G418 is selected from one or more of the following: 497.3→380.3; 497.3→338.3; 497.3→205.1; the quantitative ion pair of G418 is 497.3→163.1; (3) The collision energy for the ion pair 497.3→380.3 is 10~20 eV; the collision energy for the ion pair 497.3→338.3 is 10~20 eV; the collision energy for the ion pair 497.3→205.1 is 20~30 eV; the collision energy for the ion pair 497.3→163.1 is 20~40 eV.

16. The method of claim 15, wherein the method comprises one or more of the following features: (1) Declustering voltage is 130 V; (2) The collision energy for the ion pair 497.3→380.3 is 16 eV; the collision energy for the ion pair 497.3→338.3 is 16 eV; the collision energy for the ion pair 497.3→205.1 is 24 eV; and the collision energy for the ion pair 497.3→163.1 is 28 eV.

17. The method of any one of claims 1 to 3, wherein the method comprises one or more of the following features: (1) The inner diameter of the chromatographic column in the liquid chromatography is 1 to 8 mm; (2) The length of the chromatographic column in the liquid chromatography is 50 to 300 mm; (3) The filler particle size of the chromatographic column in the liquid chromatography is 0.5~8μm; (4) The chromatographic column in the liquid chromatography is a Waters ACQUITY UPLC Glycan BEH Amide chromatographic column; (5) Drying gas flow rate is 5~15L / min; (6) Drying gas temperature is 200~500℃; (7) Atomizing gas pressure is 20~50psi; (8) Sheath gas temperature is 200~500℃; (9) Sheath gas flow rate is 6-18 L / min; (10) Capillary voltage is +1500V to +3500V; (11) Nozzle voltage is +300V to +700V; (12) Detector gain voltage is 150~250V; (13) The acceleration voltage of the collision cell is 2~6V; (14) The dwell time is 50~150ms.

18. The method of claim 17, wherein the method comprises one or more of the following features: (1) The inner diameter of the chromatographic column in the liquid chromatography is 1 to 5 mm; (2) The length of the chromatographic column in the liquid chromatography is 150 mm; (3) The particle size of the filler in the chromatographic column of the liquid chromatography is 1.7 μm; (4) Drying gas flow rate is 8~12L / min; (5) Drying gas temperature is 300~400℃; (6) Atomizing gas pressure is 40~45psi; (7) Sheath gas temperature is 300~400℃; (8) Sheath gas flow rate is 10-15 L / min; (9) Capillary voltage is +2000V to +3000V; (10) Nozzle voltage is +400V to +600V; (11) The detector gain voltage is 200 V; (12) The acceleration voltage of the collision cell is 4 V; (13) The dwell time is 80~120ms.

19. The method of claim 18, wherein the method comprises one or more of the following features: (1) The inner diameter of the chromatographic column in the liquid chromatography is 2.1 mm; (2) Drying gas flow rate is 9~11L / min; (3) Drying gas temperature is 330~350℃; (4) Atomizing gas pressure is 35 psi; (5) Sheath gas temperature is 330~370℃; (6) Sheath gas flow rate is 11-12 L / min; (7) Capillary voltage is +2500V; (8) Nozzle voltage is +500V; (9) The dwell time is 100 ms.

20. The method of claim 19, wherein the method comprises one or more of the following features: (1) Drying gas flow rate is 10L / min; (2) Drying gas temperature is 350°C; (3) Sheath gas temperature is 350°C; (4) Sheath gas flow rate: 12 L / min; (5) The chromatographic column in the liquid chromatography has the following specifications: Waters ACQUITY UPLC Glycan BEHAmide Column 130Å 2.1mm×150mm, 1.7μm.

21. The method of any one of claims 1-3, wherein the method uses a standard addition method to detect the sample.

22. The method of any one of claims 1-3, wherein the sample is subjected to ultrafiltration prior to detection.

23. The method of claim 22, wherein the molecular weight cut-off of the ultrafiltration operation is 5 to 20 KD.

24. The method of claim 22, wherein the molecular weight cut-off of the ultrafiltration operation is 10 KD.

25. Use of the method according to any one of claims 1 to 24 in detecting G418 in a biological product or an intermediate product produced during its preparation or purification.

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