Measurement setup: temperature cycling

Thermal cycling on Prometheus Panta enables direct measurement of protein refolding capacity after temperature stress. This provides critical data for formulation optimization, developability assessment, and process parameter definition.
This article outlines setup and data interpretation for a Thermal Cycling experiment using Trastuzumab, a monoclonal IgG, as an example protein.

Method Principle & Setup

**Figure 1.** Measurement parameters for a Temperature Cycling experiment in PR.Panta Control with 5 mg/mL Trastuzumab in PBS. The blue line indicates the temperature profile that will be applied for the measurement.

In a Temperature Cycling experiment, the First Temperature is the baseline temperature at which the protein should refold after each cycle, its value is typically set to room temperature (25°C).

The Second Temperature is the first elevated temperature that the protein is challenged with. Set this value a few degrees below the onset temperature of the protein to ensure that it can fully refold during the initial cycle of your experiment (e.g., 60 °C for a stable antibody).

The First and Second Temperature Duration define how long the sample is held at each temperature per cycle. A few minutes is usually enough to observe the protein’s behavior at the elevated hold. Both Temperature Duration can be set with a minimum value of one minute.

To efficiently step through a temperature range without running excessive cycles, enable the Incremental Cycle switch and set the Temperature Increase parameter to, for example, 5°C to raise the second temperature after each cycle by that amount.

The number of Measurement Cycles should be set so that the final temperature irreversibly denatures the protein.

Please note, that for experiments with total durations > 180 min or temperatures > 95 °C, sealing of capillaries is advised in order to avoid air-bubble formation and extensive evaporation (see Capillary Sealing Paste).

Data Interpretation

**Figure 2.** Multi-parameter Temperature Cycling analysis showing the 350 nm/330 nm fluorescence ratio, turbidity, and cumulant radius as a function of time for the temperature profile set up in Figure 1 with 5 mg/mL Trastuzumab in PBS. The temperature profile is depicted as dashed lines.

In the first two cycles (60 °C and 65 °C), Trastuzumab shows small changes in the nanoDSF ratio, suggesting partial unfolding or a conformational rearrangement that affects its tryptophan fluorescence. However, turbidity and cumulant radius remain unchanged, and upon returning to 25 °C, the nanoDSF ratio fully recovers to its initial value, which confirms that unfolding is still reversible at these two temperatures.

In the third cycle (70 °C), however, the cumulant radius begins to increase and does not recover upon returning to 25 °C, indicating the onset of irreversible unfolding. The nanoDSF ratio also fails to return to its baseline value, supporting this finding.

At 75 °C, the cumulant radius exceeds 25 nm, and turbidity begins to rise, indicating the formation of larger aggregates. At 80 °C, large-scale aggregation occurs, with a sharp increase in turbidity and particle sizes in the micrometer range detected by DLS.

When lowering the Temperature Increase parameter to the minimum value of 1 °C, the reversible and irreversible unfolding behavior of a protein can be mapped with high temperature resolution (see Figure 3).

**Figure 3.** Temperature Cycling experiment with Incremental Cycle turned on and Temperature Increase set to 1°C. Dynamic light scattering was turned off. The green data points represent 1 mg/mL Trastuzumab in PBS pH 7.4, while purple is sodium acetate buffer pH 5.

Summary:

Stepwise thermal stress with return to baseline: Samples undergo controlled heating steps (e.g., 25°C → 60°C → 25°C → 65°C → 25°C → 70°C → 25°C) with conformational and colloidal assessment after each cooling phase.
Reversibility monitoring in real-time: After each temperature excursion, the protein's return to its native conformational state is tracked, distinguishing reversible thermal transitions from irreversible unfolding events.
Determination of irreversible denaturation onset: The cycling protocol identifies the temperature threshold where the protein fails to recover its initial state upon cooling, which is a key stability parameter for risk assessment.

Application Examples

Developability assessment and formulation development

Deselect candidates prone to irreversible aggregation under thermal stress
Identify constructs or conditions for reversible unfolding
Perform thermal stress studies with customizable temperature cycling and stepping

For more applications please check out the second mode within Temperature Control - Temperature Stepping.

This experiment type can be unlocked for Prometheus Panta through a separate software activation process. For more information, please contact our specialists.

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