Discussion

The assessment of protein stability is of key importance for drug development, drug discovery and basic research. Both the thermal and chemical stability of biologicals are optimized for screening campaigns, large-scale production and long-term storage. In order to meet the needs of increasing pace and competition in the design of biologicals, NanoTemper Technologies developed the Prometheus NT.48 instrument. Thermal and chemical stability of proteins can be now monitored at unmatched speed and precision using our advanced Differential Scanning Fluorimetry, nanoDSF. The hallmark of the Prometheus NT.48 is the label-free, high-throughput fluorimetric analysis of protein folding. Upon chemically and thermally induced unfolding changes in the fluorescent properties of the amino acid tryptophan are detected. Measurements are performed in 48 capillaries in parallel, require just 10 µL of sample and detect a broad dynamic protein concentration range from 5 µg/mL to 150 mg/mL.
Here we present three thermal unfolding examples. First, using antibodies we show domain-specific unfolding transition points as well as the effects of drug conjugation to antibodies. Second, we demonstrate the influence of different detergents on the stability of integral membrane proteins. These experiments highlight that nanoDSF is not affected by amphiphilic and autofluorescent additives. Finally, the high precision of the Prometheus NT.48 is demonstrated in quality control experiments. Single scan analysis precisely determined the fraction of unfolded protein in e.g. long-time stability and forced degradation tests.