Detecting DTPA content in ADC by LC-MS / MS

Abstract

The present invention relates to a method for detecting the DTPA content in an ADC by LC-MS / MS. [Effect] In this method, the sample is Fe 3+ The derivatization is performed under weakly alkaline derivatization conditions by , and at the same time, a weakly alkaline mobile phase is adopted, and no ion-pair reagent is required to be added. This method has the characteristics of high efficiency, stability, and durability, and can adequately compensate for the shortcomings of the few DTPA detection methods and complicated operation in the prior art.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting DTPA by LC-MS / MS in the field of biomedicine, which is highly accurate, durable, and reproducible. [Background technology]

[0002] Diethyltriaminepentaacetic acid (DTPA) is an aminopolycarboxylic acid consisting of a diethylenetriamine backbone with five carboxymethyl groups.

[0003] DTPA was widely used in the late 1950s to promote the excretion of radionuclides, and its calcium salt is currently the primary form. The toxic side effects of DTPA and its related calcium salts include lethal reactions at high doses, liver and kidney damage, intestinal mucosa damage, and hematopoietic inhibition (Zhao Xingcheng et al., The Toxicity and Excretion-Promoting Applications of DTPA, International Medicine (Radiation Medicine Volume), April 1980).

[0004] Currently, DTPA is widely used in the production of ADC products, being added during the reduction process of monoclonal antibodies to protect proteins after reduction. DTPA can also be used as a linker in antibody-drug conjugates (Virginia del Solar et al., Metal-based antibody drug conjugates. Potential and challenges in their application as targeted therapies in cancer, Journal of Inorganic Biochemistry 199, 2019), and is used to prepare platinum metal-antibody drug conjugates. Without DTPA, ADC production processes have shown that ADCs are not properly conjugated or the quality of the conjugated product is reduced. Existing ADC manufacturing processes typically lack a specific removal process for DTPA, and DTPA is removed only during the product dialysis process. This means that DTPA may remain in the ADC product, potentially posing a significant risk to the safety of subsequent drug use. Prior research into the detection of DTPA in ADC-related products has yet to be reported.

[0005] Currently, DTPA detection methods reported in the literature are primarily focused on the food and textile industries. Gas chromatography and high-performance liquid chromatography are often used, with ion chromatography also being employed. However, when detecting DTPA using gas chromatography, DTPA and its complexes contain polar carbonyl groups, making volatilization difficult. Therefore, esterification reactions are required to produce volatile compounds, making the process complicated. Furthermore, HPLC has a high detection limit and is not suitable for detecting residual amounts. For these reasons, LC-MSMS has attracted increasing attention. For example, Patent Document 1 (CN107315056A) discloses a method for measuring DTPA-Ca in rat plasma biological samples using LC-MSMS, which has excellent chromatographic separation efficiency and high target substance recovery. Patent Document 2 (CN107167541A) discloses a method for measuring DTPA-Zn in rat plasma biological samples using LC-MSMS, which also has excellent separation efficiency. However, these methods cannot be applied to DTPA in antibody-drug conjugate products because the antibody-drug conjugate is a mixture of protein biopolymers, making the system complex and significantly hindering the detection of DTPA.

[0006] LC-MS / MS is actually a comprehensive detection technology that combines LC and two MS technologies. It combines liquid chromatography (LC) and mass spectrometry (MS). The operating principle is that an injected sample is first carried by a mobile phase into a chromatographic column, and after separation by the chromatographic column, it is then detected in a mass spectrometer. In a mass spectrometer, detection is performed according to the mass-to-charge ratio (m / z). The analyte is converted into gas ions in the ion source, which are then detected in a mass spectrometer. The primary mass analyzer in the triple quadrupole scans a specific range of ions or allows specific ions to enter the collision cell. In the collision cell, molecular ions collide and decompose to form daughter ions, which are then detected in a secondary mass spectrometer. The secondary mass analyzer scans a specific range of ions or allows specific ions to enter the detector. The significant advantage of combining LC-MS / MS is that gas chromatography can only separate volatile, non-decomposing substances, while liquid chromatography significantly expands the separation range. The combination of LC and highly selective and sensitive MS / MS makes it possible to perform real-time analysis of complex samples. Even when separation by LC is difficult, neutral fragment scanning of the target compound by MS1 and MS2 can be performed to find and highlight the target compound in the mixture, significantly improving the signal-to-noise ratio. Summary of the Invention [Problem to be solved by the invention]

[0007] In the LC-MSMS method for detecting DTPA, Fe 3+ Derivatization methods are often used, but the derivatization conditions are always acidic, and good separation can be achieved simply by adding an ion-pair reagent to the mobile phase, making the method unstable and unreproducible. [Means for solving the problem]

[0008] In the present invention, in order to detect DTPA more quickly and effectively, a method for detecting DTPA by LC-MS / MS based on weakly alkaline derivatization conditions was developed, which effectively resolved the difficulties encountered in detecting DTPA using conventional techniques. [Effects of the Invention]

[0009] In the present invention, LC-MSMS detection is used to detect DTPA in antibody-drug conjugate products, which has high accuracy, durability, and reproducibility, and is of great application value in the quality control of ADC production. In the present invention, LC-MS / MS method is used to detect DTPA in antibody-drug conjugate products, and the sample is derivatized with FeCl3 under weakly alkaline derivatization conditions. The derivatization time is short, and the derivatized sample is stable within 8 hours. In addition, a weakly alkaline mobile phase is used, and a relatively clear chromatographic peak can be generated in a C18 chromatographic column without adding an ion-pairing reagent. This method is efficient, stable, and durable. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the linear fitting standard curve for DTPA. [Figure 2] FIG. 2 shows the detection results of DTPA content measurement by LC-MS / MS method. DETAILED DESCRIPTION OF THE INVENTION

[0011] The first aspect of the present invention is a method for detecting the DTPA content in an ADC sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS or LC-MS / MS), wherein the ADC sample to be tested contains Fe. 3+ The method is characterized in that the chromatographic column is derivatized under weakly alkaline derivatization conditions using a C18 chromatographic column and a weakly alkaline mobile phase, without the need to add an ion-pairing reagent.

[0012] Furthermore, the term "weakly alkaline" in the present invention refers to a pH range of 7≦pH≦8.

[0013] Furthermore, the Fe 3+ Derivatization with is derivatization using FeCl3.

[0014] Furthermore, the mobile phase A is an aqueous solution containing ammonium acetate and ammonium hydroxide, preferably an aqueous solution containing 80 to 100 mM of 0.2% ammonium acetate (W / V) and 0.01% ammonium hydroxide (W / V), and most preferably an aqueous solution containing 100 mM of 0.2% ammonium acetate (W / V) and 0.01% ammonium hydroxide (W / V).

[0015] Furthermore, the mobile phase B is an acetonitrile solution containing ammonium acetate and ammonium hydroxide, preferably an acetonitrile solution containing 80 to 100 mM of 0.2% ammonium acetate (W / V) and 0.01% ammonium hydroxide (W / V), and most preferably an acetonitrile solution containing 100 mM of 0.2% ammonium acetate (W / V) and 0.01% ammonium hydroxide (W / V).

[0016] Furthermore, the concentration of the FeCl3 solution to be added is about 40 to 60 μg / ml, more preferably 50.0 μg / ml.

[0017] In the method according to any one of the embodiments of the first aspect of the present invention, the standard solution is prepared using DTPA.

[0018] Furthermore, the standard solution is preferably a 1.0 μg / ml DTPA solution.

[0019] Furthermore, the preparation of the DTPA solution and the standard curve series solution is carried out as follows: 1) A step of preparing a DTPA working solution by dissolving 5-10 mg of DTPA standard weighed in an EP tube in 1 ml of 0.1% ammonium hydroxide to obtain a mother solution, and then gradually diluting the mother solution until the DTPA concentration reaches 1.0 μg / ml; 2) preparing a standard curve series solution by diluting the 1.0 μg / ml DTPA standard solution with mobile phase A to prepare DTPA standard solutions having series concentrations of 500 ng / ml, 400 ng / ml, 200 ng / ml, 100 ng / ml, and 50 ng / ml.

[0020] The method further includes using DTPA quality control solutions at concentrations of 400 ng / ml (HQC), 200 ng / ml (MQC), and 100 ng / ml (LQC), prepared by diluting a 1.0 μg / ml DTPA solution with mobile phase A.

[0021] In the method according to any one of the embodiments of the first aspect of the present invention, the liquid chromatograph includes, but is not limited to, Agilent 1200 Liquid Chromatograph, Agilent Corporation, USA, Teledyne Isco High Pressure Preparative Liquid Chromatograph, and Agilent HPLC 1260 High Performance Liquid Chromatograph.

[0022] In the method according to any one of the embodiments of the first aspect of the present invention, the tandem mass spectrometer includes, but is not limited to, an API 4000 tandem mass spectrometer manufactured by AB Sciex, USA, a triple quadrupole liquid chromatograph mass spectrometer manufactured by Shimadzu, and an Optimass inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometer manufactured by GBC.

[0023] In the method according to any one of the embodiments of the first aspect of the present invention, a C18 column is employed for liquid chromatography, including, but not limited to, YMC-C18, Agilent Extard-C18, and Shiseido PAK CR-18.

[0024] In any embodiment of any aspect of the present invention, any technical feature may be applied to the corresponding technical feature in other embodiments unless a contradiction occurs. The entire contents of all documents cited in the present invention are incorporated herein by reference, and if the meanings described in these documents are inconsistent with the present invention, the description of the present invention shall prevail. [Example]

[0025] Example 1 Measurement of DTPA content by liquid chromatography-tandem mass spectrometry (LC-MS / MS) method 1) Preparation of working fluid

[0026] Preparation of DTPA working solution: 5-10 mg of DTPA standard was weighed into an EP tube and dissolved in 1 ml of 0.1% ammonium hydroxide to obtain a mother solution, which was then serially diluted until the DTPA concentration reached 1.0 μg / ml.

[0027] Preparation of ferric chloride working solution: 10-20 mg of ferric chloride was weighed into an EP tube and dissolved in an appropriate amount of water to obtain a mother solution, which was then diluted stepwise until the ferric chloride concentration reached 50.0 μg / ml.

[0028] Preparation of standard curve series solutions: A 1.0 μg / ml DTPA (A1) standard solution was diluted with mobile phase A according to Table 2 to obtain DTPA standard solutions with concentrations of 600 ng / ml, 400 ng / ml, 200 ng / ml, 100 ng / ml, and 50 ng / ml. 200 μl of each of the above DTPA standard solutions was taken, and 5 μl of ferric chloride solution (concentration 50.0 μg / ml) was added to each. The solutions were mixed by vortexing and incubated at room temperature in the dark for 9 hours, after which detection was performed directly.

[0029] [Table 1]

[0030] Preparation of quality control solutions: 1.0 μg / ml DTPA (designated A1' and required fresh dilution from the mother liquor) standard solution was diluted with mobile phase A according to Table 2 to obtain DTPA quality control solutions with concentrations of 400 ng / ml (HQC), 200 ng / ml (MQC), and 100 ng / ml (LQC). 200 μl of each of the above DTPA quality control solutions was taken, and 5 μl of ferric chloride solution (concentration 50.0 μg / ml) was added to each. The solution was mixed by vortexing and incubated at room temperature in the dark for 4 hours, after which detection was performed directly.

[0031] [Table 2]

[0032] 2) Measurement by liquid chromatography-tandem mass spectrometry Sample preparation: The ADC sample was diluted to an appropriate concentration using mobile phase A (aqueous solution containing 100 mM 0.2% ammonium acetate and 0.01% ammonium hydroxide) as a diluent. (The DTPA content in the sample should be within the linear range. If the range is exceeded, the dilution factor must be increased or decreased and detection must be repeated.) 200 μl of the diluted sample was taken, and 5 μl of ferric chloride solution (concentration 50.0 μg / ml) was added. The mixture was mixed with a vortex and incubated at room temperature in the dark for 4 hours, after which detection was performed directly.

[0033] Preparation of mobile phase A: Weigh out 0.771 g of ammonium acetate, add 80 ml of water, and stir to dissolve. Then, add water to make the volume 100 ml and filter through a GHP membrane to obtain 100 mM ammonium acetate. 1000 μl of 100 mM ammonium acetate and 500 μl of 10% ammonium hydroxide were accurately taken, and water was added up to the 500 ml mark. Filter through a GHP membrane to obtain mobile phase A.

[0034] Preparation of mobile phase B: 1000 μl of 100 mM ammonium acetate and 500 μl of 10% ammonium hydroxide were accurately taken, and then added to 500 ml of acetonitrile. The mixture was filtered through a GHP membrane to obtain mobile phase B.

[0035] For detection, elution was performed using mobile phase A and mobile phase B according to the concentration gradient shown in Table 3.

[0036] [Table 3]

[0037] 3) Data processing and analysis Evaluation criteria: Standard curve: r≧0.9800, recovery rates at the upper and lower limit of quantification concentration points were in the range of 80.0% to 120.0%.

[0038] The quality control recoveries were in the range of 80.0% to 120.0%. At least 50% of the quality control samples at each concentration met this range, and at least four quality control concentration points from the same analytical lot met this requirement.

[0039] The specific results were as follows:

[0040] [Table 4]

[0041] The standard curve obtained after linear fitting was as follows: Y=16058x-416744, R 2 =0.9936 According to the results in Figure 1, R 2 >0.99, indicating good fitting.

[0042] Example 2 Results and evaluation of detection method 1) Durability (stability)

[0043] Durability as a function of derivatization temperature: 200 μl of DTPA standard solution with a concentration of 600 ng / ml was taken, and 5 μl of ferric chloride solution (concentration: 50.0 μg / ml) was added, mixed with a vortex, and allowed to react in a dark place for 4 hours. The influence of the derivatization temperature on the results was investigated when the derivatization temperatures were room temperature, 37°C, and 50°C, respectively. The specific results are shown in Table 5.

[0044] [Table 5]

[0045] Durability as a function of derivatization time: 200 μl of 600 ng / ml DTPA standard solution was taken, and 5 μl of ferric chloride solution (concentration: 50.0 μg / ml) was added. The mixture was mixed with a vortex and allowed to react in the dark at room temperature. The derivatization time was then carried out for 4, 6, and 8 hours, respectively, to investigate the effect of the derivatization time on the results. The specific results are shown in Table 6.

[0046] [Table 6]

[0047] Stability of the measured solution (sample for precision test): After DTPA was derivatized by reacting with ferric chloride, the solution was left in the sample tray for 0, 4, and 8 hours to examine the change in the content of the standard DTPA. The specific results are shown in Table 7.

[0048] [Table 7]

[0049] Evaluation criteria: concentration (relative standard deviation RSD%)≦25.0%. From the experimental results in Tables 5 to 7, the RSD% was less than 25.0%, which indicates that this method has strong durability.

[0050] 2) Reproducibility After the instrument was stabilized, three standard solutions (DTPA concentration 200ng / ml) were derivatized according to the experimental method, injected and analyzed, and the measurement results of six solutions were evaluated. The results are shown in Table 8. The chromatograms were collected, and the content RSD of the main peak was calculated.

[0051] [Table 8]

[0052] In the field of quantitative analysis, reproducibility refers to the precision of results obtained by the same analyst under the same operating conditions within a relatively short time interval. The standard for acceptable reproducibility is a main peak concentration RSD of 15.0% or less. Since the RSD in this test was <15.0%, this method was found to have good reproducibility, with an RSD much smaller than the highest ratio.

[0053] The above has detailed the spirit of the present invention through the preferred embodiments of the present invention. Those skilled in the art will understand that any modifications made to the above embodiments according to the technical essence of the present invention, as well as equivalent changes and modifications, shall fall within the protection scope of the present invention.

Claims

1. A method for detecting the content of diethyltriaminepentaacetic acid (DTPA) in an ADC (antibody-drug conjugate) by liquid chromatography-tandem mass spectrometry (LC-MS / MS), in which the ADC sample to be tested is subjected to weak alkaline derivatization conditions to detect Fe. 3+ and separating the resulting product by liquid chromatography and detecting it by tandem mass spectrometry. In the separation step, a C18 chromatographic column is used, and a weakly alkaline mobile phase including mobile phase A, which is an aqueous solution containing ammonium acetate and ammonium hydroxide, is used, and there is no need to add an ion-pairing reagent to the weakly alkaline mobile phase.

2. The Fe 3+ Derivatization with FeCl 3 2. The method of claim 1, wherein the derivatization is carried out using

3. 2. The method of claim 1, wherein the mobile phase A is an aqueous solution containing 80-100 mM of 0.2% ammonium acetate and 0.01% ammonium hydroxide.

4. 2. The method of claim 1, wherein the mobile phase A is an aqueous solution containing 100 mM 0.2% ammonium acetate and 0.01% ammonium hydroxide.

5. 2. The method of claim 1, wherein the weakly alkaline mobile phase comprises a mobile phase B that is an acetonitrile solution containing ammonium acetate and ammonium hydroxide.

6. The method of claim 5, wherein the mobile phase B is an acetonitrile solution containing 80-100 mM of 0.2% ammonium acetate and 0.01% ammonium hydroxide.

7. 6. The method of claim 5, wherein the mobile phase B is a solution of 100 mM 0.2% ammonium acetate and 0.01% ammonium hydroxide in acetonitrile.

8. Added FeCl 3 The method according to claim 5, wherein the concentration of the solution is 40 to 60 μg / ml.

9. Added FeCl 3 6. The method of claim 5, wherein the concentration of the solution is 50.0 μg / ml.

10. The method according to claim 8 or 9, characterized in that the standard solution is prepared by employing DTPA.

11. 11. The method of claim 10, wherein the standard solutions are prepared based on a 1.0 μg / ml DTPA working solution.

12. The standard solution is a standard curve series solution, The preparation of the DTPA working solution and the preparation of the standard curve series solutions 1) preparing a DTPA working solution by dissolving 5-10 mg of DTPA standard weighed in an EP tube in 1 ml of 0.1% ammonium hydroxide to obtain a mother solution, and then gradually diluting the mother solution until the DTPA concentration reaches 1.0 μg / ml; 2) preparing the standard curve series solutions by diluting a 1.0 μg / ml DTPA working solution with mobile phase A to prepare the standard curve series solutions having series concentrations of 500 ng / ml, 400 ng / ml, 200 ng / ml, 100 ng / ml, and 50 ng / ml.

13. The standard solution is a DTPA quality control solution, 11. The method of claim 10, further comprising using the DTPA quality control solutions at concentrations of 400 ng / ml (HQC), 200 ng / ml (MQC), and 100 ng / ml (LQC) prepared by diluting a 1.0 μg / ml DTPA working solution with mobile phase A.

Images