Measurement setup: temperature stepping

Temperature stepping on Prometheus Panta enables sequential isothermal stability assessment across multiple temperatures in a single automated experiment, streamlining formulation screening and accelerating shelf-life prediction studies.
This article outlines setup and data interpretation for a Thermal Stepping experiment using Trastuzumab, a monoclonal IgG, as an example protein.

Method Principle & Setup

**Figure 1.** Measurement parameters for a Temperature Stepping 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 Stepping experiment, the First Temperature defines the starting temperature of the experiment (e.g., 60°C). It should be chosen low enough to ensure the protein is still in its native state.

The Step Duration defines how long the sample is held at each temperature step. 20 minutes is often enough to observe whether the protein remains stable or begins to unfold at that temperature.

Set the Temperature Increase to move to the next temperature after each hold.

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

Please note, that the stepping direction can also be reversed. For this, click the thermometer icon on the right of the Temperature Increase, which changes the property to Temperature Decrease.

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 Stepping 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.

During the first two 20-minute holds (at 60 °C and 65 °C), Trastuzumab shows a constant nanoDSF ratio, as well as no increase in turbidity or cumulant radius, which indicates that it is still conformationally and colloidally stable. At 70 °C, we can observe a first slow increase of the nanoDSF ratio value, concurrent with an increase in the cumulant radius. Trastuzumab unfolds, but unfolded monomers do not yet aggregate.

At 75 °C, the cumulant radius indicates formation of larger oligomers, and particle sizes above 20-25 nm can also be spotted by the increase in turbidity.

Finally, at 80 °C, the turbidity signal significantly increases, and the cumulant radius indicates the presence of micrometer sized particles.

Summary:

Sequential isothermal holds in one experiment: Samples are maintained at defined temperatures for specified durations (e.g., 60°C for 20 min → 65°C for 20 min → 70°C for 20 min) with continuous monitoring of conformational and colloidal stability.
Real-time stability tracking at each temperature: Protein aggregation, unfolding, and colloidal changes are measured throughout each isothermal phase, providing time-resolved stability data across multiple stress conditions.

Application Examples

Formulation screening, process development and shelf-life studies

Apply defined isothermal holds to assess stability across multiple thermal stress conditions. Use it to support formulation screening and shelf-life studies across multiple stress conditions.

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

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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