A method for evaluating the stability of antibody-drug conjugates
By optimizing plasma incubation conditions, including the use of antioxidants, controlling oxygen participation and incubation pH, combined with high-resolution mass spectrometry detection, the problem of long-term stability evaluation of antibody-conjugated drugs in the systemic circulation is solved, and more accurate stability evaluation is achieved.
Patent Information
- Application Number
- CN202210263972.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-03-17
- Publication Date
- 2025-06-03
- Estimated Expiration
- 2042-03-17
AI Technical Summary
The prior art is difficult to effectively evaluate the long-term stability of antibody-conjugated drugs in the systemic circulation, especially the challenge of long-term incubating blood systems in simulated intravenous administration environments.
By optimizing plasma incubation conditions, including the use of antioxidants, controlling oxygen participation and incubation pH, combined with high-resolution mass spectrometry detection, simulating the stability changes of drugs under systemic circulation conditions, conditions suitable for long-term plasma incubation were screened out.
It effectively extends the incubation time of the blood system, improves the ability to evaluate the stability of antibody-conjugated drugs, and provides a stability evaluation system that is closer to the internal environment.
Smart Images
Figure CN115963233B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for evaluating drugs, and specifically to a method for evaluating the stability of antibody-drug conjugates. Background Art
[0002] Antibody-drug conjugates (abbreviated as ADCs) are novel drug molecules composed of antibodies linked to small molecule toxins. This type of drug generally enters the body through intravenous administration, reaches the target tissue during the systemic circulation process, the antibody binds to the antigen on the surface of tumor cells, and the linker breaks in the tumor cells, releasing the small molecule toxin. Whether the linker can break at the appropriate time and in the appropriate organ is the key to the success of ADC drugs.
[0003] Moreover, ADC drugs generally have a relatively long half-life. Whether they can stably exist during the long systemic circulation is also an indicator for evaluating whether ADC drugs will produce off-target toxicity. According to different application scenarios, the systems for evaluating the stability of ADC drugs include buffer salt systems, serum, plasma, whole blood, etc. Among them, the whole blood and plasma incubation systems are preferably used as the systems for stability investigation during drug design because they can best reflect the intravenous administration environment. However, since whole blood / plasma itself is rich in various proteins, it is not suitable for long-term incubation. And the circulation time of ADC drugs in the body is longer than several days. How to better extend the incubation time of the blood system for evaluating the stability of ADC drugs becomes particularly important. Summary of the Invention
[0004] The purpose of the present invention is to solve the above-mentioned deficiencies of the prior art. By investigating the influence of various incubation factors and optimizing the plasma incubation conditions, a method for evaluating the stability of antibody-drug conjugates is provided, and the method can better reflect the stability of ADC drugs in the systemic circulation.
[0005] To achieve the above purpose, the present invention adopts the following technical solutions.
[0006] A method for evaluating the stability of antibody-drug conjugates, comprising the following steps:
[0007] 1) Add the drug to plasma, and the final concentration of the drug is 0.1 - 1 mg / mL;
[0008] 2) Set specific conditions (using antioxidant vs not using antioxidant, passing oxygen vs not passing oxygen, incubation pH = 7.4 vs pH = 5.0), and in vitro incubate the antibody-drug conjugate with plasma to simulate the stability change of the drug under the condition that the drug contacts blood for a long time in the systemic circulation;
[0009] 3) Extract the plasma sample to obtain the small molecule metabolites generated in the plasma after incubation;
[0010] 4) Detect the samples after plasma extraction using high-resolution mass spectrometry, identify the putative drug metabolites from the mass spectrometry data, evaluate whether the culture conditions of the established incubation system can produce specific metabolites, and the level of response of the produced metabolites, in order to obtain optimized culture conditions.
[0011] Furthermore, orthogonal experimental grouping is adopted, with one single variable controlled for different groupings each time. Examine the use of antioxidants, the participation of oxygen, and the incubation pH value. Set long-term incubation (such as incubating for 14 - 30 days) to simulate the time of ADC drugs in the systemic circulation. Examine the changes in the appearance of plasma after long-term incubation under different incubation conditions (whether antioxidants are added, different incubation pH values) (such as color, protein precipitation, etc.), and through techniques such as organic solvent extraction and mass spectrometry detection, search for specific metabolites. By comparing the peak intensities of specific metabolites, evaluate the incubation effect. Only the system conditions that can detect specific predicted metabolites are considered to be the environment for normal metabolite production, and the level of intensity of specific metabolites can be used to evaluate the quality of the incubation system conditions.
[0012] Furthermore, the incubation conditions that can be used for evaluation include the use of antioxidants, the participation of oxygen, and changes in the incubation pH value. Determine the optimized incubation system conditions based on the level of response of specific metabolites obtained from mass spectrometry instrument detection. The incubation process is carried out in a CO 2 incubator, with the temperature set at 37°C, and necessary humidity (95 - 100%) can be provided to maintain the biological activity in the plasma.
[0013] Furthermore, in the method of the present invention, phosphate buffer solution with pH = 7.4 is used to dilute the plasma, and the dilution ratio is 1:1 (v:v).
[0014] The advantages of the present invention are:
[0015] 1. By examining the effects of different incubation conditions, determine the freshness preservation degree of plasma samples, and evaluate the plasma incubation effect by detecting the production amount of specific metabolites through mass spectrometry.
[0016] 2. Through comparison, screen out the conditions suitable for long-term in vitro plasma incubation.
[0017] 3. Using the method of the present invention to evaluate the stability of ADC drugs can effectively solve the problem of how to better extend the incubation time of the blood system, filling the blank of the stability evaluation system for this new type of drug, antibody-drug conjugate. Description of the Drawings
[0018] Figure 1 is the mass spectrometry signal diagram of specific metabolites used to evaluate the incubation results.
[0019] Figure 2It is the comparison result of the mass spectrometry response of the characteristic metabolite ion (m / z 447.18) under different incubation conditions. Detailed implementation manners
[0020] Example 1:
[0021] Incubate the antibody-drug conjugate (trade name: Kadcyla) in plasma with an incubation volume of 400 μL and a final incubation concentration of 1 mg / mL. Through the following different groups, compare the ability of small molecule cleavage metabolites generated under different incubation conditions.
[0022] 1. Use of antioxidants
[0023] Parallelly incubate different groups, including adding and not adding the antioxidant citric acid (10 μL, 1 mg / mL) to the plasma. Determine the improvement of diluted plasma for long-term incubation by comparing the color change and sedimentation before and after 21-day plasma incubation.
[0024] 2. Investigation of oxygen participation
[0025] Parallelly incubate different groups, including sealing the incubation tube after filling with oxygen and open incubation with daily supplementation of phosphate buffer (pH = 7.4) to the initial volume. Determine the improvement of diluted plasma for long-term incubation by comparing the color change and sedimentation before and after 21-day plasma incubation.
[0026] 3. Investigation of the pH condition of the incubation system
[0027] Parallelly incubate different groups, including diluting the plasma with phosphate buffer at pH = 7.4 or sodium acetate buffer at pH = 5.0 in a 1:1 (v:v) ratio. Determine the improvement of diluted plasma for long-term incubation by comparing the color change and sedimentation before and after 21-day plasma incubation.
[0028] 4. After the incubation of each group above is completed, precipitate and extract the plasma with 3 volumes of acetonitrile, and detect specific metabolites by high-resolution mass spectrometry. Evaluate the incubation effect by comparing the peak intensities of specific metabolites.
[0029] Results:
[0030] Figure 1 The provided mass spectrometry detection results are used to evaluate whether the incubation system is successful, where the signal of the target molecule is m / z 447.1797. By comparing the signal levels of this target metabolite molecule, the effects of different incubation conditions are evaluated. Table 1 summarizes the signal values of this target molecule under different incubation conditions.
[0031] Table 1
[0032]
[0033] After 21 days of incubation under different incubation conditions, the plasma incubated in each group underwent sedimentation to varying degrees. Among them, due to the open environment and incubation at 37°C, the water evaporation rate was relatively fast, and it was necessary to add phosphate buffer in a timely manner. The protein sedimentation was obvious. It was not suitable for the operation of high-throughput incubated samples.
[0034] The addition of the antioxidant citric acid would inhibit the activity of certain enzymes, and the content of its metabolites was lower than that of the group without citric acid.
[0035] No expected metabolites were produced during the plasma incubation under acidic conditions. Attention should be paid to the control of the pH value (controlled between 7 and 8) when selecting plasma.
[0036] Conclusion:
[0037] After 21 days of incubation under different incubation conditions, after extraction with organic solvents, the characteristic metabolite M1 (m / z 447.18) was detected using high-resolution mass spectrometry. As Figure 2 shown, the comparison results are as follows:
[0038] It was determined through comparison that the system without citric acid and the condition of pH = 7.4 were more suitable for the production of characteristic cleavage products and could be applied to evaluate the stability of antibody-drug conjugates in plasma.
Claims
1. A method for evaluating the stability of an antibody-drug conjugate, characterized in that, it comprises the following steps: 1) Add the antibody-drug conjugate to plasma, and the final concentration of the drug is 0.1-1 mg / mL; 2) Set specific conditions, including: the use of antioxidants, the participation of oxygen, and the change of incubation pH value. Incubate the antibody-drug conjugate with plasma in vitro to simulate the stability change of the drug under the condition of the drug contacting blood for 14-30 days in the systemic circulation. The specific conditions include the following several groups of comparison conditions: using antioxidants and not using antioxidants, passing in oxygen and not passing in oxygen, and incubation pH = 7.4 and pH = 5.0; 3) Extract the plasma sample to obtain the small molecule metabolites generated in the plasma after incubation; 4) Use a high-resolution mass spectrometry instrument to detect the sample after plasma extraction, identify and speculate on the drug metabolites from the mass spectrometry data, evaluate whether the culture conditions of the established incubation system can produce specific metabolites, and the level of response of the generated metabolites, so as to obtain optimized culture conditions.
2. The method for evaluating the stability of an antibody-drug conjugate according to claim 1, characterized in that, it comprises the following steps: Adopt orthogonal experimental grouping, control 1 single variable for different groupings each time, investigate the use of antioxidants, the participation of oxygen, and the incubation pH value, set long-term incubation to simulate the time of the antibody-drug conjugate in the systemic circulation, investigate the changes in the appearance of the plasma after long-term incubation under different incubation conditions, and search for specific metabolites. By comparing the peak intensities of the specific metabolites, evaluate the incubation effect. Only the system conditions that can detect specific predicted metabolites are considered to be the environment where metabolism is normally generated, and the level of the intensity of the specific metabolites can be used to evaluate the quality of the incubation system conditions.
3. The method for evaluating the stability of an antibody-drug conjugate according to claim 1, characterized in that, The incubation is carried out in a CO 2 incubator with the temperature set at 37°C.
4. The method for evaluating the stability of an antibody-drug conjugate according to claim 1, characterized in that, it comprises the following steps: Dilute the plasma with a phosphate buffer solution with pH = 7.4.