CN115290766A - Method for accurately and rapidly determining content of 2-deoxy-2-fluoro-L-fucose in antibody drug - Google Patents
Method for accurately and rapidly determining content of 2-deoxy-2-fluoro-L-fucose in antibody drug Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
The invention discloses a method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody drug, which comprises the steps of respectively adding ethanol into a matrix standard solution and a sample to be detected of the antibody drug, shaking up and oscillating, carrying out protein precipitation and then carrying out solid-liquid separation to respectively obtain a sample loading solution of the standard solution and a sample loading solution of the sample to be detected, and then analyzing by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer by a standard curve method to further obtain the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug. The method for determining the content of the 2-deoxy-2-fluoro-L-fucose in the antibody medicament has the advantages of simple operation, high analysis speed and good accuracy.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody medicament.
Background
2-deoxy-2-fluoro-L-fucose is an L-fucose analog that can be converted intracellularly to GDP-2-deoxy-2-fluoro-L-fucose, a competitive alpha-1, 3-fucosyltransferase inhibitor. It can reduce fucosylation of IgG in the antibody, enhance antibody-dependent cytotoxicity, and thereby increase the therapeutic effect of the antibody. However, studies have shown that 2-deoxy-2-fluoro-L-fucose is also an active drug that can be used in combination with checkpoint inhibitors to effectively treat cancer. In order to prevent the influence on the therapeutic effect of the target antibody drug, the residue needs to be completely removed in the purification process.
No literature report is available on the content detection of 2-deoxy-2-fluoro-L-fucose, and only a few detection methods are available on L-fucose. Fucose belongs to monosaccharide, has no absorption of ultraviolet and visible light, and is generally determined by gas chromatography and high performance liquid chromatography. The detection of the fucose can be realized by the gas chromatography only by the derivation of the fucose, and the steps are complicated. The high performance liquid chromatography is mainly divided into two types: direct assays and pre-column derivatization, in which high performance liquid chromatography without derivatization is performed using an Evaporative Light Scattering Detector (ELSD) or a differential Refractometer (RID). The differential refraction detector has higher requirement on the stability of the environment temperature, is not suitable for gradient elution analysis, and has low sensitivity and long balance time; the sensitivity of the evaporative light scattering detector is slightly higher than that of the RID, but the sensitivity is still not ideal, the baseline noise is large, and the repeatability is poor; and the high performance liquid chromatography of pre-column derivatization adopts an ultraviolet detector for detection, so that the steps are complicated, and the blank interference is large.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody drug aiming at the defects in the prior art, and the method is simple to operate, high in analysis speed and good in accuracy.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody medicament comprises the following operation steps:
(1) Preparing a matrix standard solution; the sample to be detected is a liquid antibody drug preparation, or the antibody drug is prepared into a solution;
(2) Adding ethanol into the matrix standard solution and the sample to be detected respectively, shaking up and oscillating, carrying out solid-liquid separation after protein precipitation, and obtaining a loading solution of the standard solution and a loading solution of the sample to be detected respectively;
(3) Analyzing the sample loading liquid of the standard solution obtained in the step (2) by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer to obtain a chromatogram, and establishing a linear standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate;
(4) And (4) under the same condition as the step (3), analyzing the sample loading solution of the sample solution to be detected obtained in the step (2) by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer to obtain a chromatogram, and substituting the peak area into the linear standard curve obtained in the step (3) to calculate the content of 2-deoxy-2-fluoro-L-fucose in the sample to be detected, namely the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug.
According to the scheme, in the step (1), the concentration of the standard substance in the matrix standard solution is 5-100 mu g/L. The preparation method of the matrix standard solution comprises the following steps: preparing a standard substance stock solution of the 2-deoxy-2-fluoro-L-fucose standard substance by using water, and then diluting the standard substance stock solution by using a substrate until the concentration of the 2-deoxy-2-fluoro-L-fucose is 5 mu g/L-100 mu g/L; the matrix is a monoclonal antibody pharmaceutical preparation without 2-deoxy-2-fluoro-L-fucose.
According to the scheme, the sample to be detected is generally a monoclonal antibody pharmaceutical preparation sold in the market or to be sold in the market, is usually in a liquid state, and is directly used as a sample loading solution after protein precipitation.
According to the scheme, in the step (2), the ethanol is pre-cooled ethanol with the temperature of-80-0 ℃; when ethanol is mixed with a matrix standard solution (or a sample solution to be detected), the volume ratio of the ethanol to the matrix standard solution is 1; the oscillation time is 1-3 min; the solid-liquid separation adopts a centrifugal mode, the centrifugal speed is 8000-15000 rpm, and the centrifugal time is 5-10 min.
According to the scheme, in the step (3) and the step (4), the ultra-high performance liquid chromatography is reversed-phase chromatography; the triple quadrupole mass spectrometry conditions are electrospray ionization, negative ion scanning mode, and Multiple Reaction Monitoring (MRM) methods.
Preferably, the triple quadrupole mass spectrometry conditions are: electrospray ionization, negative ion scanning, 2-3.0L/min of atomizing gas, 0-10L/min of drying gas, 5-10L/min of heating gas, 200-300 ℃ of ion source interface temperature, 150-300 ℃ of desolventizing tube temperature and 300-400 ℃ of heating module temperature.
Preferably, the ultra high performance liquid chromatography conditions are: the chromatographic column is C18 (100mm x 2.1mm I.D.,1.9 μm), the mobile phase A is a mixed solution of water and formic acid, and the phase B is a mixed solution of acetonitrile and formic acid, and gradient elution is carried out. Wherein, the volume ratio of water to formic acid in the phase A is 9995-9999, and the volume ratio of acetonitrile to formic acid in the phase B is 9995-9999.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention firstly utilizes the ultra-high performance liquid chromatography and triple quadrupole mass spectrometry to detect the content of 2-deoxy-2-fluoro-L-fucose in the antibody pharmaceutical preparation. The analysis of the carbohydrate compounds generally has the defects of no reservation in a reversed phase chromatographic system, low ionization efficiency of mass spectrometry, incomplete fragment information and the like, and generally improves the ionization efficiency of the chromatographic reservation and the mass spectrometry and the accuracy of fine structure analysis by a derivatization method, but the derivatization step is complicated. Furthermore, derivatization is more difficult to achieve with trace amounts of 2-deoxy-2-fluoro-L-fucose remaining in the antibody preparation. 2-deoxy-2-fluoro-L-fucose is greatly interfered by a matrix of an antibody pharmaceutical preparation in mass spectrometry. The invention develops a mass spectrometry method which does not need derivatization, has high analysis speed, small matrix interference, high sensitivity and good repeatability.
(2) In triple quadrupole mass spectrometry, a multiple reaction monitoring mode (MRM) adopted by the invention performs collision induced dissociation on the selected specific parent ions, removes interference of other daughter ions, only acquires mass spectrum signals of the selected specific daughter ions, has high sensitivity, and can effectively eliminate false positive phenomenon.
(3) The invention has the advantages of less sample sampling amount, simple and quick pretreatment, simple protein precipitation and centrifugation, and no need of complex treatment steps, and can obviously improve the sensitivity of subsequent detection. However, in the present invention, when eliminating matrix interference, the method for pre-treating protein precipitation is critical, and the temperature and the proportion of ethanol cannot be changed at will.
In conclusion, the method for detecting the content of the 2-deoxy-2-fluoro-L-fucose in the antibody pharmaceutical preparation is established by pretreating a sample by a protein precipitation method, is simple and quick, and is combined with ultra-high performance liquid chromatography and triple quadrupole mass spectrometry. The method is simple and rapid, has high sensitivity and good repeatability, and has important significance for detecting the content of 2-deoxy-2-fluoro-L-fucose in an antibody pharmaceutical preparation and controlling the safety of the quality of the medicine.
Drawings
FIG. 1 is a MRM chromatogram of 5. Mu.g/L matrix standard solution in example.
FIG. 2 is a matrix calibration curve in the examples.
FIGS. 3 to 6 are MRM chromatograms of samples 1 to 4 of the antibody pharmaceutical preparations in the examples in sequence.
Detailed Description
For a better understanding of the present invention, the following examples are given to further illustrate the present invention, but the present invention is not limited to the following examples.
In the following examples, 2-deoxy-2-fluoro-L-fucose as a standard was commercially available from pharmaceutical companies, ethanol and acetonitrile were obtained from Honeywell corporation, formic acid was obtained from Shanghai' an spectral laboratory science and technology Co., ltd, and water was Yibao purified water.
In the following examples, a 2-deoxy-2-fluoro-L-fucose-free pharmaceutical preparation of monoclonal antibodies as a substrate and a pharmaceutical preparation of monoclonal antibodies as a sample to be tested, wherein the monoclonal antibody drugs consist of two light chains and two heavy chains having a molecular weight of about 150000Da, the two light chains and the two heavy chains consisting of 20 amino acids arranged in this order, were prepared.
In the following examples, a system combining Shimadzu ultra high performance liquid chromatograph LC-30A and triple quadrupole mass spectrometer LCMS-8050 was used. The specific configuration is as follows: LC-30AD multiplied by 2 infusion pump, DGU-20A5 online degasser, SIL-30AC autosampler, CTO-20AC column incubator, CBM-20A system controller, LCMS-8050 triple quadrupole mass spectrometer, labsolutions Ver.5.99 chromatographic workstation. The analysis conditions were as follows:
conditions of the ultra-high performance liquid chromatograph: the chromatographic column is a reversed phase chromatographic column, the flow rate is 0.3mL/min, the sample injection amount is 5 mu L, the mobile phase A is an aqueous solution with the volume fraction of 0.05 percent formic acid, the phase B is an acetonitrile solution with the volume fraction of 0.05 percent formic acid, and the specific gradient elution procedure is shown in Table 1;
TABLE 1 gradient elution procedure
Triple quadrupole mass spectrometer conditions: electrospray ionization, negative ion mode, 3.0L/min of atomizing gas, 10L/min of drying gas, 10L/min of heating gas, 300 ℃ of an ion source interface, 150 ℃ of a desolventizing tube and 400 ℃ of a heating module.
Examples
A method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody medicament comprises the following steps:
(1) Preparation of matrix Standard solution
Weighing 1mg of commercially available 2-deoxy-2-fluoro-L-fucose, preparing 1mg/mL standard substance stock solution by using water, and diluting by using water step by step to obtain 5mg/L,10mg/L,20mg/L,50mg/L and 100mg/L standard working solutions respectively. 2 μ L of standard working solution with each concentration is taken respectively, 198 μ L of matrix (the matrix is a monoclonal antibody pharmaceutical preparation without 2-deoxy-2-fluoro-L-fucose) is added to prepare linear standard curve, and matrix standard solutions with the concentrations of 5 μ g/L,10 μ g/L,20 μ g/L,50 μ g/L and 100 μ g/L are obtained respectively.
(2) The sample solution to be tested is the antibody pharmaceutical preparation sample No. 1, no. 2, no. 3 and No. 4 produced by a certain company in a liquid state.
(3) Pretreatment of matrix standard solution and sample solution to be tested
Respectively and accurately sucking 50 mu L of matrix standard solution with each concentration or a sample to be detected into a plastic centrifuge tube, respectively adding 150 mu L of ethanol precooled at the temperature of 20 ℃, shaking for 3min, and centrifuging for 5min at the rotating speed of 15000rpm to respectively obtain the sample loading liquid of the standard curve solution with each concentration and the sample loading liquid of the sample to be detected.
TABLE 2 MRM parameters
* Representing a quantitative ion pair.
(4) And (3) analyzing the sample loading solution of the standard curve solution obtained in the step (3) by adopting an ultra-high performance liquid chromatography and a triple quadrupole mass spectrometer (the MRM parameter of the 2-deoxy-2-fluoro-L-fucose is shown in table 2) to obtain a chromatogram, and establishing a linear standard curve (shown in figure 2) by taking the concentration of the standard curve solution as a horizontal coordinate and the peak area as a vertical coordinate.
As shown in figure 1, in the MRM chromatogram of the matrix standard solution of 5 mug/L in the example, the 2-deoxy-2-fluoro-L-fucose chromatographic peak pattern is good, and no matrix interference exists at the peak position. As shown in FIG. 2, the concentrations in the examples are 5. Mu.g/L, 10. Mu.g/L, 20. Mu.g/L, 50. Mu.g/L and 100. Mu.g/L, respectively, and the matrix standard curves have good linearity and the correlation coefficient is larger than 0.9999.
(5) And (5) under the same condition as the step (4), analyzing the sample loading solution of the sample to be detected obtained in the step (3) by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer to obtain a chromatogram, and substituting the peak area into the linear standard curve obtained in the step (3) to calculate the content of the 2-deoxy-2-fluoro-L-fucose in the antibody drug.
As shown in FIGS. 3 to 6, in the MRM chromatograms of the antibody pharmaceutical preparation samples in the examples, 2-deoxy-2-fluoro-L-fucose was not detected in the antibody pharmaceutical preparation samples 1 and 2, and the contents of 2-deoxy-2-fluoro-L-fucose measured in the antibody pharmaceutical preparation samples 3 and 4 were 5.3. Mu.g/L and 23.5. Mu.g/L, respectively.
In order to investigate the accuracy and precision of the method for rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug, a standard recovery experiment is carried out. The method comprises the following specific steps: 2-deoxy-2-fluoro-L-fucose was added to antibody pharmaceutical preparation sample No. 1 at concentrations of 10. Mu.g/L and 50. Mu.g/L, respectively, and after pretreatment according to the above step (3), measurement was carried out according to step (5), and the measurement was repeated 6 times, and the average measurement results are shown in Table 3.
TABLE 3 results of recovery test with spiked standard (n =6, concentration unit. Mu.g/L)
As can be seen from Table 3, the recovery rate of 2-deoxy-2-fluoro-L-fucose is 97.3% -100.8%, RSDs is not more than 6.61%, which shows that the method for rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in antibody drugs, which is established by the invention, has high accuracy and good reproducibility.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.
Claims (7)
1. A method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody medicament is characterized by comprising the following operation steps:
(1) Preparing a matrix standard solution; the sample to be detected is a liquid antibody drug preparation or an antibody drug solution;
(2) Adding ethanol into the matrix standard solution and the sample to be detected respectively, shaking up and oscillating, carrying out solid-liquid separation after protein precipitation, and obtaining a loading solution of the standard solution and a loading solution of the sample to be detected respectively; wherein, the ethanol is pre-cooled ethanol with the temperature of-80 ℃ to 0 ℃; when ethanol is mixed with a matrix standard solution or a sample to be detected, the volume ratio of the ethanol to the matrix standard solution to the sample to be detected is 1;
(3) Analyzing the sample loading liquid of the standard solution obtained in the step (2) by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer to obtain a chromatogram, and establishing a linear standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate;
(4) And (3) under the same condition as the step (3), analyzing the sample loading liquid of the to-be-detected sample solution obtained in the step (2) by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer to obtain a chromatogram, and substituting the peak area into the linear standard curve obtained in the step (3) to calculate the content of 2-deoxy-2-fluoro-L-fucose in the to-be-detected sample, namely the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug.
2. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug as claimed in claim 1, wherein in the step (1), the concentration of the standard substance in the matrix standard solution is 5 to 100 μ g/L.
3. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in an antibody drug according to claim 1, wherein the preparation method of the matrix standard solution comprises the following steps: preparing a standard substance stock solution of the 2-deoxy-2-fluoro-L-fucose standard substance by using water, and then diluting the standard substance stock solution by using a substrate until the concentration of the 2-deoxy-2-fluoro-L-fucose is 5 mu g/L-100 mu g/L; the matrix is an antibody pharmaceutical preparation without 2-deoxy-2-fluoro-L-fucose.
4. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody medicament as claimed in claim 1, wherein in the step (2), the oscillation time is 1-3 min; the solid-liquid separation adopts a centrifugal mode, the centrifugal speed is 8000-15000 rpm, and the centrifugal time is 5-10 min.
5. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody medicine according to claim 1, wherein in the step (3) and the step (4), the ultra-high performance liquid chromatography is reversed phase chromatography; the triple quadrupole mass spectrometry conditions comprise electrospray ionization, a negative ion scanning mode and a multi-reaction monitoring method.
6. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug as claimed in claim 5, wherein the conditions of the triple quadrupole mass spectrometry are as follows: electrospray ionization, negative ion scanning, 2-3.0L/min of atomizing gas, 0-10L/min of drying gas, 5-10L/min of heating gas, 200-300 ℃ of an ion source interface, 150-300 ℃ of a desolventizing tube and 300-400 ℃ of a heating module.
7. The method for accurately and rapidly determining the content of 2-deoxy-2-fluoro-L-fucose in the antibody drug according to claim 5, wherein the ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is C18, the mobile phase A is a mixed solution of water and formic acid, and the mobile phase B is a mixed solution of acetonitrile and formic acid, and gradient elution is carried out.
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| CN106645483A (en) * | 2016-12-26 | 2017-05-10 | 大连工业大学 | Method for quantitatively detecting sea cucumber polysaccharide |
| CN109900833A (en) * | 2019-04-25 | 2019-06-18 | 中国农业科学院蔬菜花卉研究所 | The ultra performance liquid chromatography tandem mass spectrum detection method of trehalose-6-phosphate content in tomato |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645483A (en) * | 2016-12-26 | 2017-05-10 | 大连工业大学 | Method for quantitatively detecting sea cucumber polysaccharide |
| CN109900833A (en) * | 2019-04-25 | 2019-06-18 | 中国农业科学院蔬菜花卉研究所 | The ultra performance liquid chromatography tandem mass spectrum detection method of trehalose-6-phosphate content in tomato |
Non-Patent Citations (1)
| Title |
|---|
| 岛津企业管理（中国）有限公司: "岛津高效液相色谱联合三重四极杆质谱测定抗体药物中2-脱氧-2-氟-L-岩藻糖的残留", 《仪器信息网HTTPS://WWW.INSTRUMENT.COM.CN/APPLICATION/SOLUTION-938009.HTML》, pages 1 - 2 * |
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