A method for detecting antibiotics in a biological sample

Abstract

This application belongs to the field of analytical chemistry, and particularly relates to a method for detecting antibiotics in biological samples. This method uses liquid chromatography-mass spectrometry for detection, and has good specificity, accuracy, sensitivity, reliability and stability, and the detection limit and quantification limit are relatively low. It can be used to detect antibiotics (such as G418) in biological samples or biological products (such as cetuximab products) or intermediate products generated during their preparation or purification process.

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 drug safety and compliance.

[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 the cells to grow in the 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 the cells that have been successfully transfected and integrated with the target gene are screened out by G418, so as 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 injection solutions 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 the liquid phase method is relatively high. For example, the ultra performance liquid chromatography - evaporative light scattering detection (UPLC - ELSD) method reported by Zhang Xufan et al. ("Establishment of an ultra 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) has a detection and quantification limit of 0.01 mg / mL for G418, which is much higher than the safe PDE value of G418 in human intravenous injection solutions and fails to 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, convenient, has good specificity, accuracy, sensitivity, reliability and stability, and has low detection limit and quantification limit.

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

[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, due to the relatively complex sample matrix, 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 protein - based matrix to the ion source 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 for example, about 80% (v / v).

[0030] In some embodiments, 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, 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, mobile phase B is an acetonitrile - water system containing a buffer salt and acids. In some embodiments, mobile phase B is an acetonitrile - water system containing ammonium formate and formic acid.

[0033] In some embodiments, the concentration of ammonium formate in 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 in 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, 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 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.

[0040] In some embodiments, the gradient elution program 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 mass spectrometry detection of G418 is 497.3 (m / z).

[0056] In some embodiments, the product ions for the mass spectrometry detection of G418 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, has 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 in their preparation and / or purification processes, and the detection limit is even 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 besides 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 options. Any examples following "for example" or "such as" are not meant to be exhaustive or limiting.

[0076] The present application includes the following embodiments:

[0077] Embodiment 1. A method for detecting G418 in a sample, characterized in that the method comprises using liquid chromatography - mass spectrometry (LC - MS) for detection, and the chromatographic column in the liquid chromatography is a hydrophilic chromatographic column.

[0078] Embodiment 2. The method according to Embodiment 1, wherein the chromatographic column is selected from silica gel columns, diol columns, amino columns, cyano columns and amide columns, preferably an amide column.

[0079] 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;

[0080] Preferably, the buffer salt is selected from: formates, such as ammonium formate; acetates, such as ammonium acetate; phosphates, such as sodium phosphate or potassium phosphate;

[0081] The acid is trifluoroacetic acid, formic acid, acetic acid or phosphoric acid;

[0082] The organic solvent is selected from one or more of acetonitrile, methanol and tetrahydrofuran, preferably acetonitrile.

[0083] 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;

[0084] Preferably, the concentration of ammonium formate in mobile phase A is 0.001-0.3 mol / L, such as 0.01-0.1 mol / L, such as about 0.03 mol / L, and the concentration of formic acid in mobile phase A is 0.01-0.5% (v / v), preferably 0.1-0.3% (v / v), such as about 0.2% (v / v);

[0085] The concentration of ammonium formate in mobile phase B is 0.001-0.3 mol / L, such as 0.01-0.1 mol / L, such as about 0.03 mol / L; the concentration of formic acid in mobile phase B is 0.01-0.5% (v / v), preferably 0.1-0.3% (v / v), such as about 0.2% (v / v);

[0086] The acetonitrile-water system contains 60-100% (v / v) acetonitrile, such as 70-90% (v / v), and again such as about 80% (v / v).

[0087] 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;

[0088] More preferably, the following gradient elution program is used:

[0089]

[0090] 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;

[0091] Preferably, it includes one or more of the following features:

[0092] (1) The declustering voltage is 100 - 160 V, such as 120 - 140 V, such as about 130 V;

[0093] (2) The precursor ion for G418 mass spectrometry detection is 497.3 (m / z);

[0094] (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;

[0095] (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.

[0096] Embodiment 7. The method according to any one of Embodiments 1 - 6, wherein the method comprises one or more of the following features:

[0097] (1) 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;

[0098] (2) The length of the chromatographic column in the liquid chromatography is 50 - 300 mm, such as about 150 mm;

[0099] (3) 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;

[0100] (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;

[0101] (5) The drying gas flow rate is 5 - 15 L / min, such as 8 - 12 L / min, such as about 9 - 11 L / min, such as about 10 L / min;

[0102] (6) The drying gas temperature is 200 - 500 °C, such as 300 - 400 °C, such as about 330 - 350 °C, such as about 350 °C;

[0103] (7) The nebulizing gas pressure is 20 - 50 psi, such as 40 - 45 psi, such as about 35 psi;

[0104] (8) The sheath gas temperature is 200 - 500 °C, such as 300 - 400 °C, 330 - 370 °C, such as about 350 °C;

[0105] (9) The sheath gas flow rate is 6 - 18 L / min, such as 10 - 15 L / min, such as 11 - 12 L / min, such as about 12 L / min;

[0106] (10)The capillary voltage is from +1500 V to +3500 V, such as from +2000 V to +3000 V, such as approximately +2500 V;

[0107] (11)The nozzle voltage is from +300 V to +700 V, such as from +400 V to +600 V, such as approximately +500 V;

[0108] (12)The detector gain voltage is 150 - 250 V, such as approximately 200 V;

[0109] (13)The collision cell acceleration voltage is 2 - 6 V, such as approximately 4 V;

[0110] (14)The dwell time is 50 - 150 ms, such as 80 - 120 ms, such as approximately 100 ms.

[0111] 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.

[0112] Embodiment 9. The method according to any one of Embodiments 1 - 8, wherein the method uses the standard addition method to detect the sample.

[0113] Embodiment 10. The method according to any one of Embodiments 1 - 9, wherein the sample is ultrafiltered before detection, and preferably the cut-off molecular weight of the ultrafiltration operation is 3 - 100 KD, such as 5 - 20 KD, such as approximately 10 KD.

[0114] Embodiment 11. Use of the method according to any one of Embodiments 1 - 10 for detecting G418 in a biological product (such as a cetuximab product) or an intermediate product generated during its preparation or purification.

[0115] The embodiments of the present invention are 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 techniques or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For the 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.

[0116] Instruments and materials used in the embodiments:

[0117]

[0118] Note: The AEX test sample stock solution refers to the solution system obtained after the anion exchange (AEX) chromatography step in the preparation process 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 in the preparation process of biological products such as cetuximab; the UF test sample stock solution refers to the solution system obtained after the ultrafiltration (UF) step in the preparation process of biological products such as cetuximab; the AC test sample stock solution refers to the solution system obtained after the activated carbon treatment (AC) in the preparation process of biological products such as cetuximab.

[0119] Example 1 Mass Spectrometry Conditions

[0120] 1.1 Establishment of precursor ions

[0121] Instrument: Agilent1290 - Agilent 6470

[0122] Chromatography 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 5min.

[0123] Mass spectrometry conditions: Ion source: AJS - ESI; Monitoring mode: MS2 SCAN; Dry gas flow rate: 10L / min; Dry gas temperature: 350°C; Nebulizer gas pressure: 35psi; Sheath gas temperature: 350°C; Sheath gas flow rate: 12L / min; Declustering voltage: 135V; Capillary voltage: +2500V; Starting mass (m / z): 300; Ending mass (m / z): 500.

[0124] Reference stock solution: Weigh 1.556mg of G418 and add water to 25ml.

[0125] Inject the reference stock solution for analysis, and obtain the precursor ion (m / z) of G418 as 497.3.

[0126] 1.2 Establishment of declustering voltage value

[0127] Monitoring mode: MS2 SIM; Precursor ion: 497.3; Declustering voltage: 10V - 200V, step size: 10V. Other conditions are the same as 1.1.

[0128] Inject the reference stock solution for analysis, and obtain the optimal declustering voltage of G418 as 130V.

[0129] 1.3 Establishment of product ions

[0130] Monitoring mode: Product Ion; MS2 scan range: 30 - 500; CE: 5eV, 15eV, 25eV, 35eV; Desolvation voltage: 130V; Other conditions are the same as 1.1.

[0131] Inject the reference stock solution for analysis, and the product ions (m / z) of G418 obtained are 380.3, 338.3, 205.1, and 163.1.

[0132] 1.4 Determination of collision energy (CE) value

[0133] Monitoring mode: Multiple reaction monitoring (MRM); CE optimization range: 2 - 60eV, step size: 2eV; Other conditions are the same as 1.1.

[0134] Inject the reference stock solution for analysis to obtain the optimal CE value, as shown in Table 1 for details.

[0135] Table 1 Mass spectrometry parameters of G418

[0136]

[0137] Example 2

[0138] Prepare the reference solution: Weigh an appropriate amount of G418 and dilute it with the basal medium solution. Then analyze this reference solution under the following conditions.

[0139] Chromatographic conditions: Chromatographic column: Waters ACQUITY UPLC Glycan BEH Amide Column 130Å 2.1mm × 150mm, 1.7μm; Mobile phase A: 0.03mol / L ammonium formate solution (containing 0.2% formic acid); Mobile phase B: 80% acetonitrile solution of 0.03mol / L ammonium formate (containing 0.2% formic acid); Column temperature: 50°C; Injection volume: 5μl; Flow rate: 0.6ml / min; The gradient program is shown in Table 2 as follows:

[0140] Table 2 Elution program

[0141]

[0142] Mass spectrometry conditions: Ion source parameters: Ion source: AJS - ESI; Scan mode: MRM; Dry gas flow rate: 10L / min; Dry gas temperature: 350°C; Nebulizing gas pressure: 35psi; Sheath gas temperature: 350°C; Sheath gas flow rate: 12L / min; Capillary voltage: +2500V; Nozzle voltage: +500V; Detector gain voltage: 200V; Collision cell acceleration voltage: 4V; Dwell time: 100ms; Collision energy (CE), etc. are shown in Table 3 below.

[0143] Table 3 Mass Spectrometry Parameters

[0144]

[0145] See the test results in Figure 1 . The results showed that under the above conditions, G418 had good retention and response.

[0146] Example 3

[0147] Liquid phase conditions:

[0148] Chromatographic column: Waters ACQUITY UPLC Glycan BEH Amide Column 130Å 2.1mm×150mm, 1.7μm; Mobile phase A: 0.03mol / L ammonium formate solution (containing 0.2% formic acid); Mobile phase B: 80% acetonitrile solution of 0.03mol / L ammonium formate (containing 0.2% formic acid); Column temperature: 50°C; Injection volume: 10μl (AC solution system), 5μl (AEX, CEX, UF solution system); Injector temperature: 5°C; Flow rate: 0.6ml / min; Gradient program see Table 4.

[0149] Table 4 Elution Program

[0150]

[0151] Mass spectrometry conditions: Ion source: AJS-ESI; Scan type: MRM; Dry gas flow rate: 10L / min; Dry gas temperature: 350°C; Nebulizing gas pressure: 35psi; Sheath gas temperature: 350°C; Sheath gas flow rate: 12L / min; Capillary voltage: +2500V; Nozzle voltage: +500V; Detector gain voltage: 200V; Collision cell acceleration voltage: 4V; Dwell time: 100ms; Collision energy (CE), etc. see Table 5 below.

[0152] Table 5 Mass Spectrometry Parameters

[0153]

[0154] Sample preparation:

[0155] Test solution stock solution: including AEX test solution stock solution, CEX test solution stock solution, UF test solution stock solution, AC test solution stock solution.

[0156] Reference stock solution: Weigh about 0.27mg of G418 → 10ml; 0.5ml → 10ml; 1ml → 20ml (basic medium solution, about 46.7ng / ml).

[0157] 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). After mixing, take an appropriate amount and place it in an ultrafiltration centrifugal tube (10 KD). Centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0158] Blank test sample solution: Take 0.8 ml of the test sample stock solution without G418 → 2 ml (basic culture medium solution). After mixing, take an appropriate amount and place it in an ultrafiltration centrifugal tube (10 KD). Centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0159] Test sample solution: Take 0.8 ml of the test sample stock solution → 2 ml (basic culture medium solution). After mixing, take an appropriate amount and place it in an ultrafiltration centrifugal tube (10 KD). Centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0160] 100% recovery solution: Take 0.8 ml of the test sample stock solution + 0.8 ml of the reference stock solution → 2 ml (basic culture medium solution). After mixing, take an appropriate amount and place it in an ultrafiltration centrifugal tube (10 KD). Centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0161] Injection analysis and results:

[0162] Take each blank test sample solution, reference solution, test sample solution, and 100% recovery solution for injection analysis. If there is a peak in the test sample solution chromatogram with the same retention time as G418 in the reference solution chromatogram, calculate it by the external standard method based on the peak area.

[0163] Calculation formula:

[0164]

[0165] Where: W S is the weighed amount of the reference substance (mg); V T is the sampling volume of the test sample (ml)

[0166] D S and D T are the dilution factors of the reference substance and the test sample, respectively;

[0167] A S is the peak area after subtracting the test sample peak area from the reference substance peak area;

[0168] A T is the test sample peak area; p is the reference substance content.

[0169] The results show that there is no interference in each blank test sample solution at the G418 peak position, among which Figure 4 exemplarily shows the detection chromatogram of the blank AEX test sample solution, Figure 5The detection chromatogram of the reference solution is shown exemplarily; and the recovery rates of the samples added in 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%.

[0170] Example 4 Linearity and Range

[0171] Sample Preparation:

[0172] Reference Stock Solution: Weigh approximately 0.27 mg of G418 → 10 ml; 0.5 ml → 10 ml (basic medium solution, approximately 934 ng / ml).

[0173] Linear Solution: Take an appropriate amount of the reference stock solution and dilute it with the basic medium solution to prepare a series of linear stock solutions. Respectively take 0.8 ml of the series of linear stock solutions, place them in 2-ml volumetric flasks, add 0.8 ml of each test sample stock solution respectively, then dilute to the mark with the basic medium solution, mix well, take appropriate amounts respectively, place them in ultrafiltration centrifuge tubes (10 KD), centrifuge at 15,000 rpm for 10 min, and take the lower-layer clear liquid.

[0174] Using the LC-MS detection conditions in Example 3, inject samples for analysis from low concentration to high concentration. Under each solution system, calculate the regression curve and correlation coefficient based on the concentration and the corresponding G418 peak area. The results are shown in Table 6 below.

[0175] Table 6 Linearity and Range

[0176]

[0177] The results show that under each solution system, the correlation coefficient r of G418 is greater than 0.990, and the intercept deviation is relatively small, all less than 10%.

[0178] Example 5 Detection Limit and Quantification Limit

[0179] According to the test results of Example 4, determine the detection limit concentration with a signal-to-noise ratio ≥ 3 and the quantification limit concentration with a signal-to-noise ratio ≥ 10. The results are shown in Table 7 below.

[0180] Table 7 Investigation Results of the Detection Limit and Quantification Limit of the G418 Detection Method under Each Solution System

[0181]

[0182] Note: The limit is 46.7 ng / ml.

[0183] The results show that under each solution system, the quantification limit of G418 is less than 30% of the limit concentration, and the detection limit is less than 20% of the limit concentration.

[0184] Example 6 Accuracy and Precision

[0185] Stock solution of reference substance: Take an appropriate amount of G418 and dilute it with the basal medium solution to about 46.7 ng / ml.

[0186] Reference substance solution: Take 0.8 ml of the stock solution of reference substance + 0.8 ml of the test substance stock solution → 2 ml (basal 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 clear liquid.

[0187] Blank test substance solution: Take 0.8 ml of the test substance stock solution without G418 → 2 ml (basal 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 clear liquid.

[0188] Test substance solution: Take 0.8 ml of the test substance stock solution → 2 ml (basal 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 clear liquid.

[0189] 50% recovery solution: Take 0.8 ml of the test substance stock solution + 0.4 ml of the stock solution of reference substance → 2 ml (basal 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 clear liquid.

[0190] 100% recovery solution: Take 0.8 ml of the test substance stock solution + 0.8 ml of the stock solution of reference substance → 2 ml (basal 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 clear liquid.

[0191] 150% recovery solution: Take 0.8 ml of the test substance stock solution + 1.2 ml of the stock solution of reference substance → 2 ml (basal 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 clear liquid.

[0192] Take each blank test substance solution, reference substance solution, test substance solution and each recovery solution, inject them for analysis, and the results are shown in Table 8.

[0193] Table 8 Investigation results of the accuracy and precision of the G418 detection method under each solution system (the base is not detected)

[0194]

[0195] The results showed that in each solution system, when G418 at a concentration level of 50% - 150% of the limit concentration was added to the test solution, the recoveries were all between 85% and 115%, and the RSD (n = 9) was all < 15%.

[0196] Example 7 Solution Stability

[0197] Sample Preparation:

[0198] Reference Stock Solution: Weigh approximately 0.27 mg of G418 → 10 ml; 0.5 ml → 10 ml; 1 ml → 20 ml (basic culture medium solution, approximately 46.7 ng / ml).

[0199] 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 15,000 rpm for 10 min, and take the lower clear liquid.

[0200] 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 15,000 rpm for 10 min, and take the lower clear liquid.

[0201] 100% Recovery Solution: Take 0.8 ml of the test 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 (10KD), centrifuge at 15,000 rpm for 10 min, and take the lower clear liquid.

[0202] Inject and analyze the reference solution and the recovery solution in each solution system after being placed at 5°C for different times. The results are shown in Table 9.

[0203] Table 9 Results of the Investigation on the Solution Stability of the G418 Detection Method in Each Solution System

[0204]

[0205] Results: In the reference solution and the test solution, when investigated at 5°C for 8 - 11 h, there was no obvious change in the peak area of G418 compared with that at 0 h (the peak area change rate was all < 20%), indicating that the reference solution and the test solution were stable under the corresponding investigation time at 5°C.

[0206] Example 8 Method Durability

[0207] The preparation of the reference solution, the test solution, and the recovery solution for the durability investigation in each system was the same as in Example 7.

[0208] Under the basic LC-MS conditions of Example 3, the liquid phase and mass spectrometry conditions were slightly adjusted respectively, and then injection analysis was carried out. The results are shown in Table 10.

[0209] Table 10 Durability test results of G418 detection method in various solution systems

[0210]

[0211] Results: After fine-tuning the relevant chromatographic and mass spectrometric parameters, the blank test solutions in each solution system did not interfere with the G418 determination, and there was no significant difference in the recoveries (RSD<15%, n=5), indicating that the method was robust.

[0212] Example 9

[0213] A sample was tested using the LC-MS detection conditions of Example 3, and the detected amount of G418 in the sample was 3.6 ng / ml. Figure 6 The detection spectrum of the sample is shown as an example.

[0214] Comparative Example 1

[0215] The reference substance stock solution of Example 1 was analyzed by injection using conditions similar to those of Example 2, except that the following chromatographic conditions were used.

[0216] 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 time: 5 min.

[0217] Test results see Figure 2 The results showed that G418 had almost no retention. In samples with complex matrices, such as inorganic salts, which do not retain the matrix, using this chromatographic condition is prone to matrix effects and contamination of the ion source.

[0218] Comparative Example 2

[0219] The reference substance stock solution of Example 1 was sampled and analyzed under conditions similar to those of Comparative Example 1, except that the following gradient elution conditions were used.

[0220]

[0221] Test results see Figure 3 The results showed that G418 had almost no reservations.

[0222] Comparative Example 3

[0223] The test solution was tested using the LC-MS detection conditions of Example 3, except that the test solution was diluted and directly injected into the sample for analysis without ultrafiltration.

[0224] During the detection process, it was found that the pressure of the chromatographic column increased significantly, indicating that the chromatographic column was blocked and the detection could not be completed.

[0225] Comparative Example 4

[0226] The test 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 by 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. However, the interference caused by the matrix can be effectively avoided by using the standard addition method as shown in Example 3.

[0227] Table 11 Results of the investigation of the spiked recovery of G418 in each solution system by the external standard method (the substrate was not detected in all cases)

[0228]

[0229] 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, can be replaced by a technical feature that achieves the same, equivalent, or similar purpose, unless otherwise specified. Therefore, unless otherwise specified, each technical feature disclosed in the present invention is only an example of an equivalent or similar technical feature 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 includes using the standard addition method and detecting by liquid chromatography-mass spectrometry (LC-MS). Ultrafiltration is performed on the sample before detection. The chromatographic column in the liquid chromatography is an amide-based hydrophilic chromatographic column. The mobile phase used in the liquid chromatography is a mixture of mobile phase A and mobile phase B. 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. The sample is a cetuximab product or an intermediate product generated during its preparation or purification process.

2. The method according to claim 1, wherein the concentration of ammonium formate in mobile phase A is 0.001 - 0.3 mol / L, and the concentration of formic acid in mobile phase A is 0.01 - 0.5% (v / v). The concentration of ammonium formate in mobile phase B is 0.001 - 0.3 mol / L; the concentration of formic acid in mobile phase B is 0.01 - 0.5% (v / v). The acetonitrile-water system contains 60 - 100% (v / v) of acetonitrile.

3. The method according to claim 2, wherein the concentration of ammonium formate in mobile phase A is 0.01 - 0.1 mol / L, and the concentration of formic acid in mobile phase A is 0.1 - 0.3% (v / v). The concentration of ammonium formate in mobile phase B is 0.01 - 0.1 mol / L; the concentration of formic acid in mobile phase B is 0.1 - 0.3% (v / v). The acetonitrile-water system contains 70 - 90% (v / v) of acetonitrile.

4. The method according to claim 2, wherein the concentration of ammonium formate in mobile phase A is 0.03 mol / L, and the concentration of formic acid in mobile phase A is 0.2% (v / v). The concentration of ammonium formate in mobile phase B is 0.03 mol / L; the concentration of formic acid in mobile phase B is 0.2% (v / v). The acetonitrile-water system contains 80% (v / v) of acetonitrile.

5. The method according to claim 1, wherein the liquid chromatography method uses 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% - 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.

6. The method according to claim 5, wherein the following gradient elution program is adopted: 。 7. The method according to claim 1, wherein the mass spectrometry uses an electrospray ionization source ESI and performs mass scanning in the positive ion multiple reaction monitoring mode MRM.

8. The method according to claim 7, wherein the method comprises one or more of the following features: (1) The declustering voltage is 100 - 160 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); (4) The collision energy is 5 - 40 eV.

9. The method according to claim 8, wherein the method comprises one or more of the following features: (1) The declustering voltage is 120 - 140 V; (2) 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; (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.

10. The method according to claim 9, wherein the method comprises one or more of the following features: (1) The 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; the collision energy for the ion pair 497.3→163.1 is 28 eV.

11. The method according to claim 1, 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 - 8 mm; (2) The length of the chromatographic column in the liquid chromatography is 50 - 300 mm; (3) The particle size of the packing material of the chromatographic column in the liquid chromatography is 0.5 - 8 μm; (4) The chromatographic column in the liquid chromatography is a Waters ACQUITY UPLC Glycan BEH Amide chromatographic column; (5) The drying gas flow rate is 5 - 15 L / min; (6) The drying gas temperature is 200 - 500 °C; (7) The nebulizing gas pressure is 20 - 50 psi; (8) The sheath gas temperature is 200 - 500 °C; (9) The sheath gas flow rate is 6 - 18 L / min; (10) The capillary voltage is +1500 V to +3500 V; (11) The nozzle voltage is +300 V to +700 V; (12) The detector gain voltage is 150 - 250 V; (13) The collision cell acceleration voltage is 2 - 6 V; (14) The dwell time is 50 - 150 ms; (15) The flow rate in the liquid chromatography is 0.2 - 1.5 ml / min; (16) The column temperature in the liquid chromatography is 20 - 60 °C.

12. The method according to claim 11, 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 - 5 mm; (2) The length of the chromatographic column in the liquid chromatography is 150 mm; (3) The particle size of the packing material of the chromatographic column in the liquid chromatography is 1.7 μm; (4) The drying gas flow rate is 8 - 12 L / min; (5) The drying gas temperature is 300 - 400 °C; (6) The nebulizing gas pressure is 40 - 45 psi; (7) The sheath gas temperature is 300 - 400 °C; (8) The sheath gas flow rate is 10 - 15 L / min; (9) The capillary voltage is +2000 V to +3000 V; (10) The nozzle voltage is +400 V to +600 V; (11) The detector gain voltage is 200 V; (12) The collision cell acceleration voltage is 4 V; (13) The dwell time is 80 - 120 ms.

13. The method according to claim 12, 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) The drying gas flow rate is 9 - 11 L / min; (3) The drying gas temperature is 330 - 350 °C; (4) The nebulizing gas pressure is 35 psi; (5) The sheath gas temperature is 330 - 370 °C; (6) The sheath gas flow rate is 11 - 12 L / min; (7) The capillary voltage is +2500 V; (8) The nozzle voltage is +500 V; (9) The dwell time is 100 ms.

14. The method according to claim 13, wherein the method comprises one or more of the following features: (1) The drying gas flow rate is 10 L / min; (2) The drying gas temperature is 350 °C; (3) The sheath gas temperature is 350 °C; (4) The sheath gas flow rate is 12 L / min; (5) The chromatographic column in the liquid chromatography has the following specifications: Waters ACQUITY UPLC Glycan BEH Amide Column 130 Å 2.1 mm × 150 mm, 1.7 μm.

15. The method according to claim 1, wherein the cut-off molecular weight of the ultrafiltration operation is 5 - 20 KD.

16. The method according to claim 1, wherein the cut-off molecular weight of the ultrafiltration operation is 10 KD.

17. Use of the method according to any one of claims 1 - 16 for detecting G418 in biological products or intermediate products generated during their preparation or purification.

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