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One-stop service for ADC bioanalysis: ELISA, ECLA, LC-MS/MS, LBA
Total antibody, conjugated antibody, free cytotoxin, immunogen analysis
ADC 
NAb 
Target 
Cytotoxin 
AntibodyBOC Sciences bioanalysis service department has a professional research team and analysis laboratory equipped with advanced instruments, as well as implemented with comprehensive information management to meet the FDA/CFDAGLP standards requirements. Our services cover pharmacokinetics, pharmacodynamics, immunogenicity and bioequivalence; we provide antibody-drug conjugates (ADCs) drugs screening and development services, as well as preclinical and clinical research to support our customers.
With the continuous progress and breakthrough of clinical research on ADC drugs, understanding the biological analysis characteristics of ADC drugs plays a vital role in their successful development, which also greatly promotes the development of biological analysis methods for ADC drugs. At the same time, the complexity of ADC molecules also brings many challenges to biological analysis. By understanding the preclinical/clinical bioanalytical parameters of ADC drugs, such as pharmacokinetic characteristics, pharmacodynamic characteristics, immunogenicity, and safety, it is helpful to comprehensively and systematically understand the mechanism and biological process of ADC drugs in the human body. It has important application value in target selection, antibody structure design, linker/small molecule toxin selection, and drug-antibody ratio (DAR) optimization. Bioanalysis will help guide the development of ADC drugs with the best safety and efficacy. The analysis of relevant parameters of ADC drugs requires mature and reliable biological analysis methods to provide key data information for preclinical/clinical studies, so as to improve the success rate of ADC drug development and clinical approval. With the accumulation of experience in biological analysis of ADC drugs, as well as the development of new analytical methods, detection platforms and technologies, the clinical application of ADC drugs has been promoted.
Bioanalysis of the ADC antibody fraction mainly focuses on the quantitative determination of plasma concentrations of total antibodies (conjugated and unconjugated forms) and ADC (conjugated form). Pharmacokinetic analysis of total antibodies mainly describes the assessment of the total content of the ADC antibody fraction and provides information to understand the evolution of antibody concentration over time. At present, the detection of total antibodies is mainly based on enzyme-linked immunoassay method (ELISA), ligand binding assay (LBA), electrochemiluminescence immunoassay (ECLA), and characteristic peptide assay (surrogate peptide). Among them, our LBA is divided into specific method or generic method. We usually prepare at least two sets of key reagents to ensure smooth completion of method development. At the same time, during method development, it is necessary to examine whether the signal response of the method to intact ADC drugs and naked antibodies conforms to the general pharmacokinetic rules. Bridging experiments should also be used to examine the consistency of the signals of naked antibodies and ADCs.
In the PK analysis of conjugated antibodies, anti-small molecule antibodies are usually used as coating reagents, and the detection antibodies are consistent with the total antibody analysis. There are many methods for measuring coupling drugs. The classical methods include ELISA, which detects ADC antibodies and coupling small molecule toxins by specific reagents (one is a reagent that binds to the antibody part, and the other is an antibody that binds to the small molecule toxin). In addition, conjugated antibodies can also be analyzed using the LBA platform and captured using anti payload antibody. At present, we have reagent stocks for popular payloads, and have established in-house methods such as MMAE, MMAF, DM1/DM4, SN-38 and DXd to meet the needs of rapid analysis. If anti-loading antibodies are not available, we can also use alternative methods for analysis of conjugated antibodies, for example, the LC-HRMS method.
The release of free small molecule toxins from ADC drugs is a serious issue in evaluating the safety and efficiency of clinical treatments, which will lead to reduced efficacy and increased risk of systemic toxicity. Therefore, detecting the content of free small molecule toxins is an important assessment in ADC bioanalysis. In most cases the concentration of free small molecule toxins is used to infer the exposure of small molecules released by ADC dissociation in the blood system. The coupling linkers of ADC drugs are usually divided into cleavable linkers and non-cleavable linkers. For different target analytes, researchers need to design different bioanalytical methods based on the characteristics of ADC drugs. The determination of free small molecule toxins can be performed using competitive ELISA method and LC-MS/MS method. The competitive ELISA method uses anti-small molecule toxin antibodies to coat microwell plates, remove proteins from the sample, mix it with labeled small molecule toxins, and finally incubate the plate to detect signals. The difficulty lies in the need to obtain anti-small molecule toxin antibody reagents. Compared with the enzyme-linked immunoassay method, the detection of the LC-MS/MS method does not require key reagents such as antibodies, and while detecting the target small molecule toxin, it can also monitor the amino acid-linker-small molecule toxin complex, broken linker-small molecule toxin complex, etc. The production and quantification of molecular complexes, small molecule multi-level metabolites, etc.
Sometimes it is necessary to further analyze the content of small molecules conjugated to the antibody. This analysis can directly provide information on ADC drug load and monitor changes in drug load. To measure small molecule analytes conjugated to ADC, our detection method uses a hybrid LBA-LC-MS/MS method, which can simultaneously measure the concentrations of total antibodies, ADC, and conjugated small molecule toxins. First capture the ADC drug through capture reagents (anti-ADC antibodies, antigens and other reagents), use enzymes to dissociate small molecule toxins (this method is suitable for ADC drugs whose linkers can be cleaved by enzymatic or chemical excision), and then separate by LC and perform MS detection. This assay can also describe the content of the ADC mixture, and changes in the concentration of the conjugated small molecule toxin can reflect the elimination of the ADC drug from the blood circulation system and the loss from the antibody backbone.
Similar to other biological macromolecule drugs, ADC drugs may also cause immunogenic reactions in subjects. ADCs may also cause immunogenicity in humans and produce corresponding antidrug antibodies (ADA) or neutralizing antibodies (Nab), which have potential impacts on pharmacokinetics, pharmacodynamics, and safety. The ADA analysis of ADC drugs may be more complicated than the ADA analysis of monoclonal antibodies. Although the main component of ADC drugs is macromolecular antibodies, the linkers and/or small molecule toxins may act as their epitopes, resulting in a more diverse ADA mechanism of anti-ADC drugs. Therefore, current multi-layer analysis strategies should be used to evaluate the immunogenicity of ADC drugs. Among them, neutralization assay is mainly used to analyze the neutralizing antibodies (NAb) of ADC. Based on this, BOC Sciences generally has two forms of neutralization assays to assess NAb levels: cell-based assays (CBA) and ligand binding-based methods (LBA). When developing CBA, it is common to use cell lines that are highly drug tolerant and that can optimize drug response, determine appropriate endpoint readings, select control NAbs, and optimize assay parameters. The LBA method has simpler experimental operations and procedures and usually has higher sensitivity, accuracy and other performance indicators.
Drug-Antibody-Ratio (DAR) represents the amount of conjugated load of the drug-antibody part of each ADC. The DAR value continuously changes with drug metabolism and is an important parameter to describe the safety and effectiveness of ADC drugs. Hydrophobic interaction chromatography (HIC) is considered to be the gold standard for the analysis of DAR values of cysteine-coupled ADC drugs, but this method requires a large amount of ADC (20-50 μg) and requires a high ADC concentration in the sample. In recent years, LC-HRMS has provided a feasible method to analyze ADC drug DAR values at the Intact and Subunit levels with its unique advantages. Compared with the HIC-UV method, the LC-HRMS method requires fewer test samples and can achieve the characterization of ADCs with different DAR values. For detailed strategies on DAR analysis, click DAR and Drug Distribution Analysis for more detailed information.
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Wang et al. performed a pioneering study in which they designed and evaluated a series of ligand-binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays for the analysis of ADC conjugation. These hybrid assays combine anti-idiotype (anti-Id), anti-payload, or universal capture reagents with cathepsin-B or trypsin digestion technology. This study aimed to evaluate the efficacy of hybrid assays as a viable alternative to LBA in ADC bioanalysis and pharmacokinetic/pharmacodynamic (PK/PD) modeling. The findings suggest that hybrid detection can serve as a valuable alternative to traditional LBA when analyzing ADCs. The researchers emphasized the importance of selecting skilled analysts, appropriate detection methods, and platforms customized for specific needs. By implementing a comprehensive strategy for ADC PK and bioanalysis from discovery to development, researchers can optimize ADC evaluation at every stage.
Fig. 1. (A) Procedure of LB-LC–MS/MS hybrid assays. (B) Hybrid assays and ligand-binding assay in antibody–drug conjugate rat PK (Bioanalysis. 2016, 8(13): 1383-401).
The purpose of this case study is to demonstrate the development and application of a novel complete quantification method for ADCs using high-resolution mass spectrometry (HRMS), enabling more complete characterization and quantification of ADCs in preclinical studies. This study used a combination of ligand binding assay (LBA) and hybrid immunocapture liquid chromatography coupled with multiple reaction monitoring mass spectrometry (LBA-LC-MRM) to quantify ADC and total antibodies. In addition, a novel intact quantitative method using LBA-LC-HRMS was developed to directly detect and quantify intact macromolecules (e.g., ADCs). The method uses immunocapture, chromatographic separation, and intact analyte detection to achieve a linear dynamic range of 1-10 μg/mL using only 25 μL plasma sample volume. This study analyzed samples from a rat pharmacokinetic (PK) study using traditional LBA-LC-MRM and a novel complete quantitative method. For naked monoclonal antibodies (mAbs), the results from both assays agreed well, demonstrating the reliability of the complete quantitative method. However, for ADC, new species were observed only using the LBA-HRMS method, suggesting that ADC has potential biotransformations not detected by traditional methods. This finding highlights the ability of a complete quantitative approach to reveal new insights into the In Vivo characterization and quantification of ADCs, providing valuable perspectives for drug candidate optimization, immunogenicity impact assessment, and safety assessment.
Fig. 2. Characterization of trastuzumab (DAR0), trastuzumab-Maia-SG3584 DAR2 (DAR2), and trastuzumab-SG3584 DAR8 (DAR8) in rat plasma using the LBA-LC-HRMS method (Anal Chem. 2021, 93(15): 6135-6144).
The analytical components that can currently be used to characterize the PK characteristics of ADC drugs include total antibodies (total antibodies, antibodies conjugated and unconjugated to small molecule toxins), conjugated antibodies (ADC, antibodies conjugated to at least one small molecule toxin, which are ADC prototype drug), free payload, conjugated payload and their analogs (payload related species, effector molecules formed after ADC is cleavage or decomposed).
The analysis of DAR value can be divided into two aspects. One is the distribution of DAR, which represents the proportion of each ADC molecule with different DAR values in the total ADC molecules; the other is the average DAR value, which is the ratio of the molar concentration of the total drug molecules and antibody molecules in the system.
ADA's analysis process includes the following four levels: screening, confirmation, titer, and neutralization experiment
a. Screening experiment: According to the screening threshold, biological samples are initially screened (there is a 5% false positive rate). Samples higher than the threshold are considered potential positive, and samples lower than the threshold are negative.
b. Confirmation test: Conduct immune confirmation on the screened positive samples to confirm the specificity between the antibody and the drug; conduct a drug addition inhibition test on potential positive samples, which can also be called a competitive inhibition test; based on the inhibition rate before and after the addition of drug to determine whether the sample is a positive sample.
c. Titer test: The titer test of ADA positive samples can analyze the strength of the antibody. The positive samples are diluted differently so that the diluted sample is the dilution multiple at the screening threshold, which is the antibody titer of ADA.
d. Neutralization experiment: Neutralizing antibody (NAb) is a special kind of ADA. NAb interferes with the In Vivo activity of the drug by blocking the product from reaching its target or interfering with receptor/ligand binding. There are currently two types of immunogenicity analyses:
1) Cell-based assay: Use cell-based in vitro functional assays to evaluate neutralizing antibodies (Nab);
2) Non-Cell-based assay: For some biological drugs, competitive ligand binding assay (LBA) can be selected to evaluate neutralizing antibodies (NAb) according to their mechanism of action.
