Assessing the Aggregation Propensity of Single-Domain Antibodies upon Heat-Denaturation Employing the ΔT_m Shift

Nano differential scanning fluorimetry is used to quantify protein thermostability and has substantially expanded the spectrum of convenient biophysical parameters used to characterize proteins. Here, this technique is used to measure the ΔT_m shift for single-domain antibodies (sdAbs), which represents a comprehensive metric for the aggregation propensity of sdAbs upon heat-denaturation. By relating two melting curves at different protein concentrations, the ΔT_m shift described in this protocol is ideally suited for high-throughput measurements to guide protein engineering, formulation development, and developability assessment of sdAbs.