Challenges of free payload concentration analysis in ADC studies

Antibody-drug conjugates (ADCs) combine the targeting precision of monoclonal antibodies with the cytotoxic potency of their payload drugs, linked together via suitable chemical linkers. This approach enables the selective delivery of therapeutic agents to tumour cells while minimising damage to healthy tissues. The selectivity reduces side effects while permitting the use of more potent chemotherapy drugs, expanding the possibilities for effective cancer treatment.

Milestones in ADC development

ADCs were first conceived in the 1960s, but a major breakthrough came in the 1970s with the advent of the hybridoma technique for producing monoclonal antibodies. This enabled lmore specific delivery of cytotoxic payloads to their targets, reducing adverse effects and enhancing efficacy. Despite these developments, the first ADC approval did not happen until 2000 with gemtuzumab ozogamicin (Mylotarg?) to treat acute myeloid leukemia. In 2013 trastuxumab emtansine was approved, the first ADC capable of targeting solid tumours. The development of more homogeneous ADCs, such as the recently approved drug T-DXd, have significantly improved the treatments for advanced breast cancer. These successes have increased interest in ADC development. By 2024, 15 ADCs received FDA approval and over 100 more were in clinical trials.

ADC pharmacokinetic evaluation

Studies involving ADCs have complex analytical requirements and biotechs and pharmaceutical companies often seek support from contract research organisations (CROs) like 91��. As covered in our previous blog post, ligand-binding assays have become the primary method for pharmacokinetic evaluations. However, there is also a strong focus on measuring free payload concentrations, the circulating toxin not bound to the antibody or the linker. Free payload concentration directly influences treatment efficacy, toxicity, side effects, and overall safety of the ADC.

Payload molecules are often derived from traditional chemotherapies or consist of small and relatively simple toxic compounds. Analytical methods for such molecules are typically well established, however adapting these methods for ADC therapies introduces challenges.

Payload measurement challenges

ADCs are designed to release minimal free payload into the systemic circulation and free concentrations are therefore low. Detecting free payload concentrations require more sensitive, innovative and sophisticated methodologies. Simple sample preparations such as protein precipitation often need to be replaced with more elaborate ones such as liquid-liquid extraction or solid phase extraction, in combination with the use of the most sensitive triple quad mass spectrometers. The newest generation of spectrometers offer a three to four-fold increase in sensitivity compared to previous models. At 91�� bioanalytical laboratories these newest spectrometers have been instrumental in detecting and quantifying the lowest payload concentrations in plasma.

Chromatographic interference

Developments of reversed-phase materials suitable for the use of highly aqueous mobile phases have enabled effective retention for even the smallest and most polar payload molecules. Large molecule ADCs, with their much bigger contact surfaces, will have much more retention and generally will not interfere on a reversed-phase liquid chromatography (LC) system. This is regardless of whether or not the molecule is left intact during sample preparation.

Column saturation affecting the payload retention is not easily achieved with one injection at circulating ADC concentrations as high as 1 mg/mL in the bloodstream. However, unless proper precautions are taken, multiple injections can lead to column-related issues over time. Incorporating a high organic flush phase in the mobile phase gradient is necessary to prevent column contamination or interference from slow moving large molecules. This flush phase will wash off the ADC and any other high retentive matrix constituents after each injection.

Free payload in ADC formulations

The ADC reference material in the formulation buffer itself will always contain some level of free payload. The manufacturer may not detect this free payload content using classical techniques like LC-UV to determine impurities. The manufacturer may conclude that impurities are below 0.1%, however, during (pre)clinical studies ADC concentrations measured in plasma from participants or testing animals are typically much higher. This discrepancy results in molar payload to ADC concentration ratios of 1:1500 or 1:30000. Therefore, even a molar impurity of only 0.035% in the reference material could lead to a 50% increase of the payload at LLOQ level. This should be considered, and where possible, corrected for in stability tests with samples spiked with both free payload and ADC.

Stability of the ADC and the payload

During sample preparation deconjugation of ADCs can significantly impact the accuracy of the measured free payload concentrations. Evaluating ADC degradation during sample preparation can be done by analysing blank matrix samples spiked with ADC at realistically high concentrations. The results can be compared to the known free payload concentrations in the ADC reference material.

The payload itself can also become unstable in plasma. 91�� has examined this phenomenon and there are indications that several common plasma additives and anticoagulants may stabilise payloads during sample preparation. We have observed that ADC deconjugation and payload degradation may occur at similar rates during sample preparation. This can lead to an inaccurate perception of ADC stability and method performance. We recommend careful examination for ADC deconjugation and payload degradation. ADC concentrations can be orders of magnitude higher than payload concentrations. Due to the excess of ADC in study samples, even the smallest amount of ADC degradation will result in huge payload biases over time.

Conclusion

Sensitive and sophisticated analysis methods are required to measure payload molecules from ADCs to measure their typically low concentrations in circulation. Payload stabilities must always be evaluated in conjunction with the ADC present in the sample. Even a small impurity or minor instability of the ADC can lead to huge changes in free payload concentrations. Innovative approaches in sample preparation, stability evaluations and chromatographic techniques will become indispensable to meet the needs of the expanding ADC developments. Ensuring accuracy and efficiency requires a deep understanding of ADC stability and payload behaviour under various conditions. Strategic optimisation of analytical workflows is essential to address the complexities of analysing ADC therapies.

This blog is adapted from the article:

91�� has extensive experience with ADC assays, including LC-MS/MS, LBA PK, and ADA methods for ADC drug development. Find out more or reach out directly.

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