During a thermal ramp of a monoclonal antibody (mAb) in Uncle, two things typically happen in tandem: the protein unfolds, shifting its intrinsic fluorescence emission to higher wavelengths, and it aggregates, increasing static light scattering (SLS). Formulations that make mAbs unfold or aggregate at lower temperatures are less stable than ones that make it break down at higher temps.
Thermal ramps, melting curves, and aggregation curves are quick and powerful protein stability assays but holding samples for a longer time at steady temperatures can paint a more detailed stability picture. Quite often, incubating a mAb at a temp below its Tagg will, eventually, induce aggregation. For example, these three formulations of the same mAb had TaggS (dotted lines) above 65 °C. However, when they were held at 60 °C for six hours, SLS increased and the mAbs aggregated in the same order as during the thermal ramp. How quickly this happens, and at what rate, is another way of ranking the stability of formulations or protein variants.