An innovative new treatment is gaining momentum in cancer therapies and giving hope to patients worldwide. Antibody-drug conjugates (ADCs) are sophisticated therapeutics that combine the targeted precision of antibodies with the potent cell-killing power of cytotoxins connected by a linker, as seen in Figure 1. ADCs deliver treatment directly to tumor cells, minimizing damage to healthy tissues and potentially transforming patient outcomes.
At the heart of Antibody-drug conjugates (ADC) technology is targeted therapy: using antibodies to seek out cancer cells with unparalleled precision. Once these antibodies bind to specific antigens on the surface of a tumor cell, they are internalized, and the cytotoxic payload is released inside the cancer cell, leading to its destruction. This mechanism enables increased efficacy and reduced toxicity.
The development and optimization of ADCs are underpinned by rigorous bioanalysis. Understanding these drugs’ metabolism and toxicity through preclinical and clinical studies is critical. The complexity of ADCs, with their variable drug-to-antibody ratios (DAR), requires sophisticated analytical techniques to quantify their components accurately. This includes measuring total antibodies, coupled antibodies (with payloads), and free payloads to fully understand the pharmacokinetics and pharmacodynamics of these drugs.
Analytical methods such as Ligand Binding Assay (LBA) and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) are at the forefront of ADC characterization.
- LBA is typically employed to quantify the total and coupled antibodies.
- LC-MS/MS offers a detailed analysis of bound and unbound cytotoxins.
These methodologies comply with and are endorsed by regulatory standards, ensuring that the development of ADCs is both scientifically rigorous and aligned with safety guidelines. Bridging the gap between antibodies’ specificity and cytotoxins’ potency allows ADCs to treat cancer more effectively and with fewer side effects.
A Synergy of Tools for Analyzing ADCs
ELISA (Enzyme-Linked Immunosorbent Assay) and ECL (Electrochemiluminescence Immunoassay) are pivotal tools in the quest for precision in therapeutic monitoring. These methodologies improve ADC evaluation by offering robust solutions to quantify total and conjugated antibodies, thus ensuring a comprehensive understanding of the drug’s behavior in the body. Common strategies for ADC bioanalysis are shown in Figure 2.
ELISA and ECL stand out for their ability to selectively quantify antibodies within biological samples. Using target antigens or monoclonal antibodies that recognize specific regions of ADCs, these assays can capture and measure the total antibodies present. These assays are also versatile enough to quantify coupled antibodies and those linked to cytotoxic payloads. They use similar capture techniques with anti-cytotoxin antibodies for detection or an alternative approach using antibodies against the cytotoxins themselves.
For instances in which ADC analysis requires a more tailored approach—particularly in detecting free cytotoxins—the preferred method becomes LC-MS/MS. This technique can handle direct analysis after standard bioanalytical pre-treatment methods, such as protein precipitation, liquid-liquid extraction, or solid-phase extraction.
However, ADC characterization can be challenging, particularly when there are prolonged preparation cycles for specific reagents and when the specificity and affinity of these reagents fall short of the ADC analysis criteria. LC-MS/MS becomes the most efficient and cost-effective solution in these scenarios. Its capability to analyze all components of the ADC in a single sample—from total antibodies to free cytotoxins—highlights its precision and unparalleled flexibility.
As we evolve our understanding of ADC development and assessment, a combination of ELISA, ECL, and LC-MS/MS methods emerges as the best path forward. Each technique brings unique strengths, but collectively, the approach offers a comprehensive toolkit for detailed analysis of ADCs.
LC-MS/MS: Full Component Analysis of ADC Drug with One Sample
The LC-MS/MS method has matured significantly in detecting protein substances, including antibodies. In analyzing ADCs, this technique is employed for the quantitative analysis of free small molecule cytotoxins and their conjugates with antibodies and for the comprehensive bioanalysis of ADC drugs. This is achieved through specific immune capture and enzymatic hydrolysis processes, which ensure excellent specificity. Particularly, the antibody component is quantitatively analyzed based on characteristic peptide fragments. Consequently, the LC-MS/MS platform allows for the simultaneous detection of total antibodies, antibody conjugates, coupled drugs, and free drugs within the same sample. The process involves obtaining the supernatant, which is then directly used to detect free cytotoxins post-immune capture. Subsequently, the captured antibody fraction is separated into two parts for individual analysis of the coupled antibody and the total antibody, as depicted in Figure 3.
Drug Analysis of Free Cytotoxins
Two critical factors in the analysis of free cytotoxins are sensitivity and stability. The concentration of free toxins in plasma is notably low, owing to the targeted nature of ADCs and the absence of a toxin-releasing tumor microenvironment, necessitating a low limit of quantitation.
As such, when the common protein precipitation approach cannot achieve the desired sensitivity, more sophisticated methods, including liquid-liquid extraction (LLE), solid-phase extraction (SPE), or supported liquid extraction (SLE), can be employed to improve assay performance. As illustrated in Figure 4, the LC-MS/MS chromatogram presents a plasma sample with 10 pg/mL MMAE in rat plasma using SLE sample preparation.
The stability of the linker connecting the antibody to the toxin in plasma is also crucial for accurately determining the free toxin’s concentration. Stability testing should assess the toxin’s stability in the biological matrix and whether the ADC releases free toxins during sample storage and pre-treatment.
Conjugate and Total Antibody Analysis
Different dissociation conditions must be selected based on the linker type to analyze antibodies’ conjugation. When using enzymes to release toxins, the choice of enzyme and the digestion conditions require careful optimization to achieve the highest possible recovery rate from enzyme digestion.
This optimization is reflected in Figure 5, where the standard curve shows good linear regression across a concentration range spanning approximately 100 folds. The appropriate enzyme cleaves cleavable linkers, allowing for direct measurement of the toxin concentration.
In contrast, for non-cleavable linkers, Trypsin or other protease can be employed to digest the protein, enabling the analysis of the toxin linker-bound portions of the antibody.
Determining the total antibody, akin to the coupled antibody, involves capture and enzyme digestion. Each step, from the selection of reagents to the reaction time and enzyme concentration, requires meticulous optimization to achieve the desired recovery rate.
Determining DAR Value
The loading of small molecule drugs on an antibody in ADCs diminishes over time due to metabolic processes, affecting the drug-to-antibody ratio (DAR). Monitoring DAR changes is crucial for understanding the safety and efficacy of ADCs. Ideally, an ADC should release the cytotoxin at the target organ rather than in the blood circulation, minimizing biological toxicity and maximizing therapeutic efficacy.
Considering that ADCs with different DARs have varying molecular masses, High-Resolution Mass Spectrometry Quadrupole-Time of Flight (HRMS-Q-TOF) analysis offers significant benefits over triple quadrupole mass spectrometry methods due to its superior resolution, specificity, and accuracy. However, HRMS-Q-TOF has limitations in sensitivity, with its lower limit of quantification reaching only the microgram per milliliter level.
DAR determination encompasses several approaches. One involves analyzing the complete ADC molecule’s DAR, requiring steps such as immune capture and deglycosylation before high-resolution mass spectrometric analysis, which directly reveals the toxin’s attachment to the ADC molecule. Alternative strategies for toxins linked specifically to light or heavy chains may involve specific reagents or enzymatic hydrolysis to analyze the ADC subunits, offering enhanced sensitivity. Tailoring these approaches to individual cases is essential for accurate analysis.
Choosing a Platform
LBA and LC-MS/MS are used to analyze total and coupled antibodies in ADC drugs, each offering distinct advantages regarding reagent specificity, sensitivity, and throughput.
The LBA platform heavily relies on the quality and preparation of specific reagents, significantly influencing the method’s specificity and development time. However, LBA excels in sensitivity, achieving picogram per milliliter-level quantitation. Conversely, the LC-MS/MS platform analyzes antibodies through characteristic peptide fragments without needing specific reagents, leading to shorter development times and lower costs compared to LBA. This benefit is particularly significant in the analysis of novel dual-antibody ADC drugs. LC-MS/ MS can also analyze various ADC forms in a single sample, with a lower limit of quantification at approximately 10 ng/mL, sufficiently meeting routine detection requirements.
When preparing specific reagents is time-consuming or their specificity is suboptimal, LC-MS/MS can facilitate rapid method development, validation, and sample analysis. It can also analyze samples within approximately six weeks, significantly reducing overall costs due to the absence of expensive reagent preparation. In addition, LC-MS/ MS offers the advantage of determining the concentration of all sample components concurrently, resulting in more comparable data. The ongoing advancement of mass spectrometry technology further enhances the sensitivity of LC-MS/MS analysis, expanding its application range.
Thus, choosing an analysis platform depends on the specific requirements of the study. If time and cost are critical factors, LC-MS/ MS is the best path forward. Working with a trusted lab partner is highly recommended if drug developers or sponsors lack the in-house capability or capacity to perform these analyses.
About WuXi AppTec
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the pharmaceutical and healthcare industry around the world to advance discoveries and deliver groundbreaking treatments to patients. Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development, and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, and cell and gene therapies CTDMO (Contract Testing, development,t, and Manufacturing Organization), helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received an AA ESG rating from MSCI in 2023 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made and every disease can be treated.”
Author Details
Pei Zhang, Associate Director of Method Development in the Bioanalysis Department- WuXi AppTec.; Wenzhong Liang, PhD, Executive Director in the Bioanalysis Department- WuXi AppTec.
Pei Zhang is the Associate Director of Method Development in the Bioanalysis Department at WuXi AppTec. She received a master’s degree in Material Science and Engineering from Soochow University in 2015. Pei has developed more than 200 molecular methods and built solid and ruggedness method development procedures for multiple new molecule modalities in her professional career. She is highly experienced and possesses extensive knowledge in the LC-MS/MS method development of small molecules, peptides, PPMO, ADCs, oligonucleotides, and other new drug modalities.
Wenzhong Liang, Executive Director in the Bioanalysis Department of WuXi AppTec, graduated from the University of Illinois at Chicago with a doctoral degree in Medicinal Chemistry. He has worked in WuXi AppTec for 12 years and has more than 20 years of experience in regulated bioanalysis and GLP laboratory management. Dr. Liang has been a standing member of the Professional Committee of Biomedical Bioanalysis of the Chinese Pharmaceutical Association since 2020.
Publication Details
This article appeared in American Pharmaceutical Review: Vol. 27, No. 4May/June 2024Pages: 78-81
Subscribe to our e-newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!