Quantitative method for detecting trace free chelate deferoxamine in ACCs medicine based on reversed phase chromatography

Abstract

The invention discloses a quantitative method for detecting trace free chelate deferoxamine in ACCs medicine based on reversed phase chromatography. The quantitative method comprises the following steps: preparing a deferoxamine standard substance solution; the method comprises the following steps: respectively detecting a sample to be detected and a deferoxamine standard substance solution by adopting a liquid chromatograph under the detection conditions that a chromatographic column is PLRP-S (2.1 * 100 mm, 5 [mu] m), the column temperature is 25-80 DEG C, and a filler is PS / DVB porous particles; mobile phases: a phase A is an aqueous solution containing organic acid, and a phase B is an organic solvent containing organic acid; establishing a calibration curve according to the concentration of the standard deferoxamine solution and the peak area of the corresponding elution peak, and then substituting the peak area of the elution peak of the sample to be detected into the calibration curve to obtain the concentration of the deferoxamine in the sample to be detected. According to the method, the tedious sample pretreatment step is omitted, the direct sample injection analysis of the sample is realized, the experimental period is greatly shortened, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a quantitative method for detecting trace free chelate deferoxamine in ACCs drugs based on reverse-phase chromatography. Background Art

[0002] Antibody-radionuclide conjugates (ARCs) are formed by conjugating a radionuclide with an antibody through a linker and a chelator to perform different functions such as in vivo diagnosis or treatment. Radionuclide targeted therapy uses a molecule highly expressed specifically in tumors as a target, and uses a targeting molecule with high affinity and specificity (such as an antibody molecule or a polypeptide molecule, etc.) to accurately deliver the radionuclide to the tumor site, and uses the rays generated by the decay of the radionuclide to exert a killing effect, performing specific and targeted internal radiation therapy on the tumor. Radionuclide targeted drugs not only kill the tumor tissues and cells on the surface or in the shallow layer of the tumor tissue through nuclear rays, but also use the penetration of nuclear rays to kill the tumor cells inside solid tumors, namely the "cross-fire" effect. Compared with pure biological drugs and chemical drugs, radioactive drugs have stronger killing power. In addition, for small-volume tumor lesions or metastases that cannot be diagnosed in time through imaging diagnosis and surgical operations, in vivo radio-targeted therapy will exert its unique efficacy.

[0003] Antibody-chelator conjugates (ACCs) are intermediate forms of ARCs, which are composed of an antibody mediating the targeting and positioning function, a linker, and a chelator. The linker and the chelator can be covalently bound to the antibody. N-hydroxysuccinimide ester (NHS) and thiocyanate (SCN) are commonly used reactive electrophilic group linkers. The linker and the chelator have a significant impact on the stability and payload of ARCs, thus affecting the drug efficacy and safety. Therefore, the residue of the chelator should be concerned during the quality research process.

[0004] p-SCN-Bn-deferoxamine (SCN-DFO) is an iron ion chelate composed of deferoxamine (DFO) and p-SCN-Bn, and its molecular formula is as follows: 。

[0005] Among them, p-SCN-Bn is an organic ligand with a specific structure, which can form stable coordination bonds with many other metal ions; DFO is a known iron ion chelate, which has the ability to form stable chelates with iron ions and is widely used for isotope chelation of gallium (Ga) and zirconium (Zr) in ARCs drugs.

[0006] Currently, the commonly used methods for the analysis and control of free chelates remaining in ACCs are as follows: 1. Liquid chromatography-tandem mass spectrometry (LC-MS / MS), which has high sensitivity and specificity, but the detection cost is relatively high and not all laboratories are equipped with mass spectrometry analysis instruments; 2. Enzyme-linked immunosorbent assay (ELISA), which is suitable for high-throughput screening or semi-quantitative analysis, but has a long development cycle, narrow application range, and lack of specificity; 3. Reverse-phase high-performance liquid chromatography-ultraviolet detection method (RP-HPLC-UV), which has high sensitivity and specificity, and the detection cost is lower than that of mass spectrometry, but it is only applicable to the detection of free chelates with ultraviolet absorption.

[0007] Among them, in the application process of the reverse-phase high-performance liquid chromatography-ultraviolet detection method, it is usually necessary to first remove the protein matrix in the sample to avoid blocking the chromatographic column and shortening the service life of the chromatographic column. However, the removal process is cumbersome, time-consuming, easily affects the detection efficiency, and also causes problems such as low extraction recovery rate of free chelates, poor reproducibility, and incomplete matrix removal, which in turn affect the chromatographic column life and chromatographic peak shape. Summary of the Invention

[0008] To overcome the above disadvantages, the purpose of the present invention is to provide a quantitative method for detecting trace free chelate deferoxamine in ACCs drugs based on reverse-phase chromatography, which omits the cumbersome sample pretreatment steps, realizes direct injection analysis of samples, greatly shortens the experimental cycle, improves the detection efficiency, and ensures the accuracy and reliability of the quantitative results.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is: A quantitative method for detecting trace free chelate deferoxamine in ACCs drugs based on reverse-phase chromatography, comprising the following steps: (1) Prepare a deferoxamine standard solution with a set concentration; (2) Use a liquid chromatograph to detect the sample to be tested and the deferoxamine standard solution respectively, and obtain the corresponding elution peak areas; wherein, the detection conditions of the liquid chromatograph are: chromatographic column: PLRP-S (2.1×100mm, 5μm), column temperature 25~80°C, packing material is PS / DVB porous particles; detector: ultraviolet detector; injection volume: 20μL; mobile phase: phase A is an aqueous solution containing organic acid, phase B is an organic solvent containing organic acid; flow rate is 0.4~0.6ml / min; detection wavelength is 290nm; (3) Establish a calibration curve based on the concentration of the deferoxamine standard solution and the corresponding elution peak area, and then substitute the elution peak area of the sample to be tested into the calibration curve to obtain the concentration of deferoxamine in the sample to be tested.

[0010] The beneficial effects of the quantitative method of the present invention are as follows: The reverse-phase chromatographic detection and analysis of the sample to be tested is achieved through the cooperation of steps (1)-(3), thereby eliminating the pretreatment process of the sample to be tested and realizing the direct injection analysis of the sample. In step (2), a chromatographic column filled with PS / DVB porous particles is used, which can directly analyze samples containing complex matrices (proteins), avoiding problems such as abnormal peak shapes and column blockages that occur when traditional C18 reverse-phase chromatographic columns analyze complex matrices. Combined with a liquid chromatograph, an ultraviolet detector, and the optimization of detection conditions, the high-efficiency analysis of the sample to be tested containing deferoxamine and its derivatives is achieved, with low detection errors, simple operation, and ensuring the accuracy and reliability of the quantitative results.

[0011] Furthermore, step (1) includes dividing a certain amount of SCN-DFO into multiple portions and diluting the multiple portions of SCN-DFO into deferoxamine standard solutions with different concentrations using a diluent; wherein, the diluent is composed of a 0.1% concentration trifluoroacetic acid acetonitrile solution and a 0.1% concentration trifluoroacetic acid aqueous solution mixed in a volume ratio of 35:65.

[0012] Furthermore, the concentrations of the standard solutions are 1 μmol / L, 2 μmol / L, 5 μmol / L, 20 μmol / L, 50 μmol / L, and 100 μmol / L respectively.

[0013] Furthermore, the pore size of the PS / DVB porous particles is 100 nm. PS / DVB (styrene / divinylbenzene) porous particles are rigid polymer particles and are suitable for the purification of small molecules, synthetic biomolecules, and macromolecules.

[0014] Furthermore, in the mobile phase, the volume ratio of the organic acid in phase A is 0.01-0.5%; the volume ratio of the organic acid in phase B is 0.01-0.5%.

[0015] Furthermore, in the mobile phase, the organic acid includes at least one of formic acid, propionic acid, lactic acid, acetic acid, or trifluoroacetic acid; the organic solvent includes at least one of methanol, ethanol, isopropanol, and acetonitrile.

[0016] Furthermore, phase A is a 0.1% concentration trifluoroacetic acid aqueous solution, and phase B is a 0.1% concentration trifluoroacetic acid acetonitrile solution.

[0017] Furthermore, in step (3), establishing the calibration curve includes: performing linear regression processing on the concentration of the deferoxamine standard solution and the corresponding elution peak area to obtain Y = a*X + b, where Y is the intensity of the ultraviolet signal; X is the concentration of the deferoxamine standard solution; a and b are constants.

[0018] Furthermore, the sample to be tested is an organic aqueous solution or protein product containing desferrioxamine. Among them, the protein in the protein product can be a polypeptide or protein for medical, pharmaceutical, or edible use. Among them, medical or pharmaceutical proteins include monoclonal antibodies, cytokines, protein hormones, enzymes, etc.

[0019] Furthermore, in step (2), the elution conditions of the mobile phase are as follows: from 0 to 5 minutes, isocratic elution with 30% B phase by volume, and the remaining 70% is A phase; from 5 to 5.1 minutes, the volume fraction of B phase linearly increases from 30% to 80%, and the volume fraction of A phase linearly decreases to 20%; from 5.1 to 7 minutes, isocratic elution with 80% B phase by volume, and the remaining 20% is A phase; from 7 to 7.1 minutes, the volume fraction of B phase linearly decreases from 80% to 30%, and the volume fraction of A phase linearly increases to 70%; from 7.1 to 15 minutes, isocratic elution with 30% B phase by volume, and the remaining 70% is A phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the HPLC chromatogram overlay of the sample processed by Method 1 in Example 1 of the present invention; Figure 2 It is the HPLC chromatogram overlay of the sample processed by Method 2 in Example 1 of the present invention; Figure 3 It is the HPLC chromatogram overlay of the sample processed by Method 3 in Example 1 of the present invention; Figure 4 It is the HPLC chromatogram overlay of the samples analyzed using different chromatographic columns in Example 2 of the present invention; Figure 5 It is the HPLC chromatogram overlay of the quantitative analysis of SCN-DFO standard solutions with different concentrations in Example 3 of the present invention; Figure 6 It is the fitting diagram of the SCN-DFO standard curve formula in Example 3 of the present invention; Figure 7 It is the HPLC chromatogram overlay of each sample in Example 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following elaborates on the preferred embodiments of the present invention in detail, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention. It should be noted that the following embodiments are for better further understanding of the present invention and are not limited to the best embodiments, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0022] It should be noted that in the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0023] In addition, for those embodiments where specific experimental steps or conditions are not specified, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. The numerical range indicated by "~" in the embodiments represents a range that includes the values recorded before and after "~" as the minimum and maximum values respectively. For the reagents or instruments whose manufacturers are not specified, they are all conventional reagent products that can be obtained through commercial purchase.

[0024] Example 1 Optimization of Sample Pretreatment (1) Sample preparation: 5 μmol / L SCN-DFO standard solution, monoclonal antibody mixture containing 5 μmol / L SCN-DFO, and ACC sample.

[0025] (2) The SCN-DFO standard solution, monoclonal antibody mixture, and ACC sample were pretreated respectively using the following methods: Method 1: 100 μL of the SCN-DFO standard solution, monoclonal antibody mixture, and ACC sample were respectively added to 300 μL of acetonitrile, vortexed for 30 s, centrifuged at 15000 rpm for 10 min, and the supernatant was taken; Method 2: 100 μL of the SCN-DFO standard solution, monoclonal antibody mixture, and ACC sample were respectively added to 500 μL of methanol, vortexed for 30 s, centrifuged at 15000 rpm for 10 min, and the supernatant was taken; Method 3: 100 μL of the SCN-DFO standard solution, monoclonal antibody mixture, and ACC sample were heated at 95 °C for 5 min, centrifuged at 15000 rpm for 10 min, and the supernatant was taken; (3) The supernatants in Method 1, Method 2, and Method 3 were respectively detected using a Waters HPLC Arc device and an InfinityLab Poroshell 120 EC-C18 chromatographic column (2.1×75 mm, 2.7 μm, Agilent) according to the detection conditions in Table 1, and the following was obtained as Figure 1-3Overlay of sample HPLC chromatograms shown.

[0026] Table 1 Test conditions

[0027] Result analysis: Figure 1 , Figure 2 The chromatograms are after protein precipitation treatment of samples with acetonitrile and methanol, respectively. It can be seen that the peak area of ​​the monoclonal antibody mixture containing 5 μmol / L SCN-DFO is significantly reduced compared with the 5 μmol / L SCN-DFO standard solution, and the recovery rate is only 60~-70%, indicating that protein precipitation treatment of samples with organic solvents will lead to the loss of SCN-DFO. Figure 3 This is the chromatogram after the sample was treated with protein precipitation by heating. It can be seen that the chromatographic peak of SCN-DFO was not detected in the monoclonal antibody mixture containing 5 μmol / L SCN-DFO, indicating that SCN-DFO is sensitive to temperature and is not suitable for high temperature protein precipitation.

[0028] In summary, no matter what method is used to precipitate the protein in the sample, the sample recovery rate will be affected, resulting in the inability to accurately quantify the trace amount of SCN-DFO in the sample. Therefore, it is necessary to directly analyze the sample by optimizing the chromatographic conditions to eliminate the complicated sample pretreatment process, improve the detection efficiency and ensure the accurate quantification of the sample.

[0029] Example 2 Optimization of chromatographic conditions When the existing C18 reverse phase chromatography column is used to directly analyze samples containing complex matrix components, especially samples containing proteins, problems such as abnormal peak shape and column clogging may occur, thereby shortening the life of the column. Therefore, this embodiment selects a reverse phase chromatography column with a larger pore size that can be used to analyze proteins.

[0030] Specifically, this embodiment uses a Water HPLC Arc device, and uses a Sepax Bio-C18 column (4.6×150 mm, 5 μm, 30 nm, Sepax) and a PLRP-S column (2.1×100 mm, 5 μm, 100 nm, Agilent), and detects a 50 μmol / L SCN-DFO standard solution according to the detection conditions in Table 2, and obtains the following Figure 4 Overlay of HPLC chromatograms of standard solutions shown.

[0031] Table 2 Test conditions

[0032] Result analysis: fromFigure 4 It can be seen that when analyzing using a Sepax Bio-C18 chromatographic column (4.6×150 mm, 5 μm, 30 nm, Sepax), there are many small impurity peaks on the right side of the chromatographic peak of SCN-DFO, indicating that the chromatographic column has poor separation effect on the impurities in the sample and the resolution of the chromatographic peak is poor; while when analyzing using a PLRP-S chromatographic column (2.1×100 mm, 5 μm, 100 nm, Agilent), there is a good resolution between the chromatographic peak of SCN-DFO and the impurity peak.

[0033] Example 3 Quantitative analysis of SCN-DFO standard solution (1) Divide an appropriate amount of SCN-DFO sample into 6 equal parts, and use a mixed solution of 0.1% trifluoroacetic acid acetonitrile solution and 0.1% trifluoroacetic acid aqueous solution in a volume ratio of 35:65 as the diluent; use the diluent to dilute the 6 parts of SCN-DFO into a series of SCN-DFO standard solutions with concentrations of 1 μmol / L, 2 μmol / L, 5 μmol / L, 20 μmol / L, 50 μmol / L, and 100 μmol / L respectively.

[0034] (2) Use a Water HPLC Arc device and a PLRP-S chromatographic column (2.1×100 mm, 5 μm, 100 nm, Agilent, PN:PL1912-2502PK), and detect different concentrations of SCN-DFO standard solutions according to the detection conditions in Table 3.

[0035] Table 3 Detection conditions

[0036] (3) Record the peak area of the elution peak at 3.7 minutes through an ultraviolet detector (UV), and process the data of the peak area of the elution peak and the concentration of the corresponding SCN-DFO standard solution.

[0037] (4) Use the formula Y = a*X + b to draw a calibration curve based on Y and X, with a weight of 1 / X; where Y is the intensity of the ultraviolet signal, representing the main peak area of SCN-DFO; X is the concentration of the SCN-DFO standard solution; a and b are constants.

[0038] Figure 5 is the superposition diagram of the detection results of different concentrations of SCN-DFO standard solutions, Figure 6 is the calibration curve for the quantitative analysis of different concentrations of SCN-DFO standard solutions. From Figure 6 it can be obtained that the elution peak area of the SCN-DFO standard solution and the corresponding concentration satisfy the following SCN-DFO standard curve formula: Y = (2.96E+04)X - (1.18E+04) Among them, Y is the intensity of the ultraviolet signal, representing the main peak area of SCN-DFO, and X is the concentration of the SCN-DFO standard solution.

[0039] It can be seen from this that the analysis result of the quantitative analysis method in this embodiment has an excellent linear relationship and accuracy. Therefore, it can be used for the quantitative analysis of SCN-DFO.

[0040] Example 4 Separation Test and Quantitative Analysis of SCN-DFO / Protein Product Mixture (1) Sample preparation: 5 μmol / L SCN-DFO standard solution, monoclonal antibody mixture containing 5 μmol / L SCN-DFO, and ACC sample conjugated with SCN-DFO.

[0041] (2) Use a Water HPLC Arc device and a PLRP-S chromatographic column (2.1×100 mm, 5 μm, 100 nm, Agilent, PN: PL1912-2502PK), and detect the above samples according to the detection conditions in Table 4.

[0042] Table 4 Detection Conditions

[0043] (3) Detect and record the chromatogram through a UV detector (UV), as Figure 7 shown, the DFO in the ACC sample conjugated with SCN-DFO elutes first and can be effectively separated from the protein.

[0044] (4) Record the peak area of the elution peak of the monoclonal antibody mixture containing 5 μmol / L SCN-DFO at 3.7 minutes, and substitute the peak area into the SCN-DFO standard curve formula in Example 3 to calculate the measured concentration of SCN-DFO in the monoclonal antibody mixture.

[0045] After analysis, when calculating the concentration of SCN-DFO in the monoclonal antibody mixture using the SCN-DFO standard curve formula, the deviation is -2.4%, meeting the error requirements. It can be proved that this method can accurately quantify SCN-DFO in protein products.

[0046] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it. It cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A quantitative method for detecting trace free chelate deferoxamine in ACCs drugs based on reverse phase chromatography, characterized in that: The steps include: (1) Prepare a deferoxamine standard solution of a set concentration; (2) Liquid chromatography was used to detect the sample to be tested and the deferoxamine standard solution, and the corresponding elution peak area was obtained; wherein the detection conditions of the liquid chromatography were as follows: chromatographic column: PLRP-S (2.1×100mm, 5μm), column temperature 25~80℃, filler: PS / DVB porous particles; detector: UV detector; injection volume: 20μL; mobile phase: phase A is an aqueous solution containing organic acid, phase B is an organic solvent containing organic acid; flow rate is 0.4~0.6ml / min; detection wavelength is 290nm; (3) A calibration curve is established based on the concentration of the deferoxamine standard solution and the corresponding elution peak area, and then the elution peak area of ​​the sample to be tested is substituted into the calibration curve to obtain the concentration of deferoxamine in the sample to be tested.

2. The quantitative method according to claim 1, characterized in that Step (1) comprises dividing a certain amount of SCN-DFO into a plurality of portions, and using a diluent to dilute the plurality of portions of SCN-DFO into deferoxamine standard solutions of different concentrations; wherein the diluent is a mixture of a 0.1% trifluoroacetic acid acetonitrile solution and a 0.1% trifluoroacetic acid aqueous solution in a volume ratio of 35:

65.

3. The quantitative method according to claim 2, characterized in that The concentrations of the standard solutions are 1 μmol / L, 2 μmol / L, 5 μmol / L, 20 μmol / L, 50 μmol / L, and 100 μmol / L, respectively.

4. The quantitative method according to claim 1, characterized in that The pore size of the PS / DVB porous particles is 100 nm.

5. The quantitative method according to claim 1, characterized in that In the mobile phase, the volume proportion of the organic acid in the phase A is 0.01-0.5%; the volume proportion of the organic acid in the phase B is 0.01-0.5%.

6. The quantitative method according to claim 1, characterized in that In the mobile phase, the organic acid includes at least one of formic acid, propionic acid, lactic acid, acetic acid or trifluoroacetic acid; the organic solvent includes at least one of methanol, ethanol, isopropanol and acetonitrile.

7. The quantitative method according to claim 6, characterized in that The phase A is a 0.1% trifluoroacetic acid aqueous solution, and the phase B is a 0.1% trifluoroacetic acid acetonitrile solution.

8. The quantitative method according to claim 1, characterized in that In step (3), establishing a calibration curve includes: performing linear regression processing on the concentration of the deferoxamine standard solution and the corresponding elution peak area to obtain Y=a*X+b, wherein Y is the intensity of the ultraviolet signal; X is the concentration of the deferoxamine standard solution; and a and b are constants.

9. The quantitative method according to claim 1, characterized in that: The sample to be tested is an organic aqueous solution or a protein product containing deferoxamine.

10. The quantitative method according to claim 1, characterized in that: In step (2), the elution conditions of the mobile phase are as follows: from 0 to 5 min, 30% of the volume fraction of phase B is eluted isocratically, and the remaining 70% is phase A; from 5 to 5.1 min, the volume fraction of phase B increases linearly from 30% to 80%, and the volume fraction of phase A decreases linearly to 20%; from 5.1 to 7 min, 80% of the volume fraction of phase B is eluted isocratically, and the remaining 20% ​​is phase A; from 7 to 7.1 min, the volume fraction of phase B decreases linearly from 80% to 30%, and the volume fraction of phase A increases linearly to 70%; from 7.1 to 15 min, 30% of the volume fraction of phase B is eluted isocratically, and the remaining 70% is phase A.

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