Antibody Drug Conjugates (ADCs), which consist of antibodies, payload and linkers, are an innovative therapeutic approach targeting various types of tumors and cancers. It can improve the therapeutic parameters of payload and reduce systemic cytotoxicity. ADCs rely on highly targeted tumor antigen recognition and effective endocytosis to recognize and bind to specific tumor antigens on the cell surface. They then enter tumor cells through endocytosis. Linkers or antibodies are dissolved in endosomes or lysosomes, releasing payload to kill tumor cells. It can prolong the half-life of payload and reduce their dose-limiting toxicity.
The biological analysis data of ADC drugs plays a vital role in the evaluation of drug metabolism and toxicity in preclinical and clinical studies. Due to the complexity of the structure of ADC drugs and the constant change of the Drug-to-Antibody Ratio (DAR) values with drug metabolism, it is necessary to quantitatively analyze several major forms of ADC drugs, including total antibodies (DAR≥0), coupled antibodies (DAR≥1), and free payload. Ligand Binding Assays (LBA) and Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) assays have been used to analyze various ADC analytes. LBA is usually used to detect total and conjugated antibodies, and LC-MS/MS is used primarily to analyze antibody-conjugated payload and unconjugated payload.
ELISA (Enzyme-Linked Immunosorbent Assay) and ECL (Electrochemiluminescence Immunoassay) are widely used for the determination of total and conjugated antibodies. Total antibodies are usually captured by the target antigen or by monoclonal antibodies in the anti-CDR region (the complementary determinant of ADC antibodies) and detected by the antibody portion of the anti-ADC drug. There are two methods for the detection of conjugated antibodies. One uses the same capture reagent as the total antibody detection, and the detection reagent is supposed to be an anti-cytotoxin antibody. Another capture reagent used is an antibody against cytotoxic drugs, and the detection reagent is a target antigen or a monoclonal antibody against the CDR region. The detection of free cytotoxin is carried out directly by LC-MS/MS after extracting the cytotoxin using pre-treatment methods commonly used in small molecule bioanalyses, such as protein precipitation, liquid-liquid extraction, or solid phase extraction.
However, when the preparation cycle of specific reagents is too long, or the specificity and affinity of reagents fail to meet the test criteria, the LC-MS/MS platform can provide a high-quality solution with fast speed and low cost and can analyze all the detected components of the ADC in one sample.
The LC-MS/MS method is gradually maturing in detecting protein substances such as antibodies. In the area of biological analysis of ADC drugs, in addition to applying it to the quantitative analysis of free small molecule payload and small molecule payload conjugated to antibodies, it is also applied to the biological analysis of ADC drugs after appropriate immune capture and enzymatic hydrolysis due to its good specificity. The surrogate peptide segment quantitatively analyzes the antibody part. This means that the same sample could be used on the LC-MS/MS platform to simultaneously detect total antibodies and conjugated and unconjugated drugs. After immune capture, the supernatant is obtained and directly used to detect free cytotoxin. The captured antibody part is divided into two parts and processed respectively to detect the conjugated drugs and total antibodies.
Drug Analysis of Free Payload
There are two important considerations for drug analysis of free payload: sensitivity and stability. The concentration of free toxin in plasma is extremely low due to the targeting nature of ADCs and the lack of toxin-releasing tumor microenvironment. Therefore, the lower limit of quantitation needs to reach the pg/mL level. In addition, since the most used protein precipitation method is supposed to interfere with the detection if the sample is not clean enough, liquid-liquid extraction or solid phase extraction is used to make the sample pre-processing cleaner and to concentrate the sample to improve sensitivity.
The plasma stability of the linker connecting the antibody to the toxin should also be investigated to obtain the true concentration of free toxin in the sample. In the stability test, it is necessary not only to investigate the stability of the toxin itself in the biological matrix but also to inspect whether the ADC releases free toxins during sample storage and processing.
Conjugate Payload Analysis
Different digestive conditions need to be selected for different linkers to analyze the conjugated payload. If enzymes are used to release toxins, the type of enzymes and the conditions of enzyme digestion must be carefully polished to obtain the highest recovery rate of enzyme digestion possible. In this way, the standard curve presents a good linear regression over a concentration range of about 100 times.
DAR Value Determination
The load of payload on the antibody will gradually decrease due to the metabolism of ADC drugs in vivo, corresponding to the change of DAR. The changing trend of DAR helps explain the safety and effectiveness of ADC. An ideal ADC drug should not release cytotoxin in the blood circulation but release it to the target organ, playing a role in reducing biological toxicity and increasing efficacy.
High-Resolution Mass Spectrometry Quadrupole-Time of Flight analyzer (HRMS-Q-TOF) shows remarkable advantages in the DAR analysis than ordinary mass spectrometry such as triple quadrupole because of its high resolution, specificity and accuracy characteristics. Still, its performance in sensitivity is poor, and its lower limit of quantification only achieves the ug/mL level for now.
The DAR value determination is divided into several cases. One is the detection of the complete ADC molecule’s DAR value. In this case, ADC molecules go through immune capture, deglycosylation and other steps and then enter the high-resolution mass spectrometry for complete molecular determination, which directly reflects the situation of the toxin attached to the ADC molecule. For some toxins only connected to light or heavy chains, it is possible to consider using specific reagents to reduce enzymatic hydrolysis and then measure the subunit of ADC, which has the advantage of higher sensitivity. This part of the program needs to be customized according to specific cases.
Total Antibody Drug Analysis
Like the conjugated antibody, the determination of the total antibody involves capture, enzyme digestion and other steps. Each step in the selection of reagents, reaction time, dosage and other aspects needs to be carefully explored to achieve the ideal recovery rate.
A Final Word
The LBA and LC-MS/MS methods analyze total antibodies and conjugated antibodies of ADC drugs, and each has its own advantages in terms of specific reagent demand, specificity, sensitivity, and flux. The LBA platform is highly dependent on the quality and preparation of specific reagents, so the quality and time of its preparation also affect the specificity and development cycle of the method, while it shows remarkable advantage in great sensitivity. The LC-MS/MS platform is used to analyze antibodies through surrogate peptide and does not rely on specific reagents, so the development cycle of the method is shorter, and the cost is lower than that of the LBA platform. This advantage is even more prominent in the biological analysis of the emerging dual-antibody ADC drugs. At the same time, one sample could be used to determine various forms of ADC; the lower limit of quantification is 10ng/mL, which perfectly meets the general detection needs. Therefore, the analysis platform could be selected according to the inspection requirements. When the preparation cycle of specific reagents is too long, or the specificity of particular reagents is not ideal, the LC-MS/MS platform for ADC drug detection could realize the rapid development and validation of methods and sample analysis within six weeks. The cost of methods and analysis will be reduced because there is no need to prepare high-cost specific reagents. In addition to the unique advantage that the LC-MS/MS platform can achieve the concentration results of all components in the same sample, the data is also better comparable. Of course, the continuous development of mass spectrometry technology gives more chances for improving the sensitivity of LC-MS/MS analysis to meet more application scenarios.
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