Ensuring ADCs’ Stability and Binding with Calorimetry

Antibody-drug conjugates (ADCs) are a cutting-edge class of biopharmaceuticals transforming oncology treatment with increased specificity and efficacy. Their impact is only growing, as 14 ADCs have received global market approval to date and hundreds more are in development.¹

ADCs successfully target and kill cancer cells with high specificity due to their hybrid makeup – they combine the selectivity of a stable, hydrophilic monoclonal antibody (mAb) with highly cytotoxic, hydrophobic small molecules. Therefore, regulatory bodies such as the FDA and EMA require hybrid characterization of ADCs to satisfy the requirements of both biological products and small-molecule drugs.²

While Mass Spectrometry has long been the standard for determining drug-to-antibody ratio (DAR), characterizing stability and binding is critical for ensuring ADCs’ safety and efficacy as well as predicting shelf-life and meeting regulatory requirements. Understanding the physical stability and binding efficacy of these complex molecules is made simple on TA Instruments’ Nano DSC and Affinity ITC. The following examples show common challenges in ADC development that can be overcome with adequate characterization.

In the process of creating ADCs, attaching the hydrophobic payloads to hydrophilic antibody molecules often disrupts the water network and secondary interactions, resulting in destabilization. Instability can cause premature release of the small molecules and lead to off-target toxicity, completely compromising the safety and efficacy of the therapy.³

Characterizing stability is made simple on the TA Instruments Nano DSC, a microcalorimeter designed to measure the molecular stability of dilute in-solution biomolecules. Figure 1 shows a Nano DSC temperature ramp of Trastuzumab (parent) versus T-DM1 (Lysine conjugate). The parent mAb is stable up to 70°C, but the ADC shows aggregation and unfolding onset as low as 45°C. This thermal shift to the left represents lower stability; this information allows developers to predict shelf-life and aggregation risks early in formulation.

Researchers from Alliance Protein Laboratories published a similar study using the Nano DSC to investigate structure and stability changes from conjugation. They saw a decrease in thermal stability when an aromatic molecule was conjugated, which was easily detectable on their Nano DSC.⁴

Formulation screening also requires testing ADCs in various commercial buffers. The Nano DSC can detect subtle shifts in the unfolding profile based on the buffer environment, helping scientists select the optimal formulation to counteract the instability caused by conjugation. For higher throughout, the TA Instruments Rapid-Screening DSC (RS-DSC) is designed to test up to 24 samples at formulation strength concentrations greater than 20 mg/mL. In the example below, the thermal stability of Herceptin Trastuzumab was tested in histidine, borate, PBS, and succinate buffer on an RS-DSC.⁵

Figure 2 and Table 1: Overlay graph of triplicate scans generated with RapidDSC using automated T_max and baseline detection of antibody thermograms in triplicate at 50 mg/mL. Detected T_max values indicated with a black dot.

The first unfolding event is not significantly impacted by the histidine, borate, or PBS buffers, but the succinate buffer destabilizes the CH2 domain resulting in lowering the onset of unfolding and T_max,1 by about 3 °C. In the main transition, the histidine and succinate buffers are the most stabilizing. Unsurprisingly, the most stabilizing buffer formulation for trastuzumab in this sample set is the histidine buffer used for final formulation of the approved drug product.

Creating ADCs involves modifying antibodies to allow for drug conjugation and to also improve tumor targeting and clearance. However, modifications to the antibody, especially to its hydrophobicity, can negatively affect its binding affinity. This challenge is especially true when using non-specific lysine conjugation near Complementarity-Determining Regions (CDRs).

Binding kinetics can be measured by Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI), but only one technique provides full thermodynamic data: Isothermal Titration Calorimetry (ITC). ITC is a direct, in-solution method that measures enthalpy, entropy, and stoichiometry of a binding event, making it the gold standard for detailed binding characterization. Furthermore, label-free detection on an ITC without immobilization eliminates artifacts that are common in SPR and BLI.

For example, the following ITC data shows repeat injections of the Trastuzumab-based antibody drug conjugate which is binding to HER2. The Trastuzumab ADC maintains a dissociation constant comparable to the parent antibody (~3 nM) and also a similar enthalpy (-96 kJ/mol). ITC is essential for confirming that any modifications to the parent antibody have not adversely affected its binding affinity or specificity.

Figure 3: ITC Binding Isotherms. A) Parent Antibody (Trastuzumab) and B) ADC binding Her2 under native conditions without immobilization, presented in this webinar.

As ADCs continue to evolve, so does their structural complexity. First-generation ADCs relied on lysine conjugation, resulting in highly heterogeneous mixtures with broad drug-to-antibody ratio (DAR) distributions ranging from 0 to 8. Second-generation approaches introduced cysteine conjugation, offering more uniform DARs (~4) but at the cost of disrupting native inter-chain disulfide bonds. Today, third-generation and dual-drug ADCs leverage site-specific conjugation using engineered cysteines or non-natural amino acids, enabling precise payload placement and controlled DARs.

With this evolution comes a new analytical challenge: predicting how these increasingly intricate architectures behave under physiological and storage conditions. While Waters LC-MS remains essential for mapping conjugation sites and calculating average DAR, it cannot fully capture the thermodynamic consequences of structural modifications. As the number of conjugation sites, payload types, and linker chemistries expands, empirical thermal profiling with Nano DSC becomes critical. By directly measuring unfolding transitions and aggregation onset, Nano DSC provides early insight into whether a novel ADC format will remain stable — or crash out of solution — under real-world conditions.

Developing successful ADCs requires a holistic approach. Complete characterization means going beyond knowing what is attached with mass spectrometry. Ensuring therapeutic efficacy and safety requires developers to assess how conjugation impacts structural stability and target binding.

As the above examples have demonstrated, Nano DSC and Affinity ITC provide the sensitive, label-free data needed to meet FDA guidelines for higher-order structure and functional assessment. DSC clarifies stability while ITC clarifies binding, and these techniques can even be combined with mass spectrometry for a more comprehensive analysis of ADC behavior.

Learn more about TA Instruments’ complete suite of biologics characterization instruments here, and contact our experts for personalized guidance in finding the right solution for your lab.

This blog was adapted from a webinar presented by Shawn C. Owen, Ph.D. for Waters TA Instruments in partnership with Bioprocess Online.

1. Fu, Z.; Li, S.; Han, S. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy 7, 93 (2022). https://doi.org/10.1038/s41392-022-00947-7
2. Clinical Pharmacology Considerations for Antibody-Drug Conjugates Guidance for Industry. FDA-2021-D-1051; Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research: 2024. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/clinical-pharmacology-considerations-antibody-drug-conjugates-guidance-industry
3. Fisher, J. E. Jr. Considerations for the Nonclinical Safety Evaluation of Antibody-Drug Conjugates. Antibodies (Basel) 10, 2 15 (2021). https://doi.org/10.3390/antib10020015
4. Arakawa, T.; Kurosawa, Y.; Storms, M.; Maruyama, T.; Okumura, C. J.; Maluf, N. K. Biophysical characterization of a model antibody drug conjugate. Drug Discoveries & Therapeutics, 10, 4 (2016). https://doi.org/10.5582/ddt.2016.01042
5. Nervig, C. Rapid Thermal Stability Screening and Selection of Monoclonal Antibody Drug Products. TA Instruments: 2024. https://www.tainstruments.com/applications-notes/rapid-thermal-stability-screening-and-selection-of-monoclonal-antibody-drug-products-mc178/