How temperature cycling experiments connect unfolding reversibility to aggregation risk.

As unfolding and aggregation are so closely linked, conformational stability and colloidal stability are therefore best evaluated together.5 Using PrometheusTM Panta, scientists can monitor thermal and colloidal stability parameters simultaneously along a linear thermal ramp. As temperature rises, nanoDSFTM tracks conformational changes to reveal unfolding (Tm), while DLS, SLS and Backreflection capture the onset temperature of size increase (Tonset rH) and formation of large amorphous aggregates (Tonset Turbidity). Together, these readouts provide a strong foundation for candidate ranking considering both thermal stability and aggregation propensity.

Why conformational and colloidal stability should be measured together.

During biologics candidate selection, thermal stability data (particularly Tm values and unfolding transitions) are routinely used to rank proteins. But a high Tm does not guarantee a candidate will resist aggregation under the milder, but prolonged or repeated stresses encountered during manufacturing and storage.1 

Even at temperatures well below their Tm, protein molecules can transiently sample partially unfolded states. When they do, exposed hydrophobic regions can promote self-interaction and aggregation. 2 Once aggregation begins, it is often irreversible: the protein is no longer free to refold. For multi-domain proteins like monoclonal antibodies, unfolding of even a single domain can be enough to trigger this cascade, posing a significant risk to both patient safety and drug efficacy.3

Therefore, characterizing whether unfolding is reversible adds critical insight beyond standard thermal ramps. It reveals whether a protein can refold after stress or instead will progress toward aggregation. As Figure 1 illustrates, the pathway from the native state can lead to either outcome, and understanding which path a candidate favors is key to assessing developability.4 

Is your protein unfolding reversible? Here's why it matters for biologics developability.

In some cases, several closely ranked candidates show similar conformational and colloidal behavior in a standard linear thermal ramp. When that happens, studying whether unfolding is reversible after thermal stress can provide an additional layer of resolution.

This can be achieved with temperature cycling experiments that capture both the thermodynamic and kinetic dimensions of unfolding reversibility. 

A temperature cycling experiment alternates the thermal profile between a baseline and a stress temperature, with the option to progressively increase the stress temperature. A practical starting point is to set the baseline temperature to 25 °C and choose an initial stress temperature a few degrees below the onset of unfolding. For a stable antibody, this might be around 60 °C. Typically, hold times of a few minutes at each temperature are applied. For a detailed walkthrough with example data, see the full temperature cycling protocol here.

By allowing protein molecules to approach thermodynamic equilibrium at both the stress and baseline temperatures, cycling makes it possible to determine:

  • Whether the protein refolds at the baseline temperature.
  • How fast refolding occurs.
  • How many cycles the protein can withstand before losing the ability to refold.
  • When aggregation begins to prevent refolding.

Temperature cycling helps identify the reversibility of unfolding threshold: the transition from recoverable stress to irreversible damage, directly informing candidate selection. 

Connect thermal stability and aggregation in one workflow.

Prometheus Panta combines nanoDSF, Backreflection, DLS, and SLS on a single instrument, simultaneously measuring both conformational and colloidal stability across flexible thermal ramps. From initial screening along a linear ramp to aggregation profiling with temperature cycling, it delivers domain-level stability data at every stage of developability.

Learn how Temperature Cycling on Prometheus Panta can help you assess reversible protein unfolding and aggregation during developability assessment.

Explore Temperature Cycling → 

Interested in Prometheus Panta for protein stability characterization?

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Why Tm is not enough: From thermal stability to aggregation risk.

Figure 1: Relationship between conformational and colloidal stability of antibodies.
Pathways from the native to the aggregated state. Domain unfolding can lead either to refolding (reversible) or to aggregation (irreversible). 

References:  

  1. Brader ML, Estey T, Bai S, et al. Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies. Mol Pharm. 2015;12(4):1005–1017. 
  2. Roberts CJ. Therapeutic protein aggregation: mechanisms, design, and control. Trends Biotechnol. 2014;32(7):372–380. 

  3. K.H. Strucksberg, T. Rosenkranz, J. Fitter. Reversible and irreversible unfolding of multi-domain proteins, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics,Volume 1774, Issue 12, 2007, Pages 1591-1603,ISSN 1570-9639, https://doi.org/10.1016/j.bbapap.2007.09.005.

  4.  NanoTemper Technologies. Biologics formulation: learn how Boehringer Ingelheim uses Prometheus to find optimal buffer conditions for their mAbs. Application Note. Link

  5. Bailly M, Mieczkowski C, Juan V, et al. Predicting antibody developability profiles through early stage discovery screening. MAbs. 2020;12(1):1743053. doi: 10.1080/19420862.2020.1743053.