Exploring Protein Stability by NanoDSF

Abstract

The assessment of protein stability is critical for drug development, drug discovery, and basic research. Both the thermal and chemical stability of biologicals, such as antibodies, are monitored to establish optimal conditions for screening campaigns, large-scale production, and long-term storage. In order to meet the needs of an increasing pace and competition in the design of biologicals and biosimilars, NanoTemper Technologies developed the Prometheus NT.48 instrument for monitoring thermal and chemical stability of proteins with an unmatched data quality, speed, and precision.The fluorescence of tryptophans in a protein is strongly dependent on its close surroundings. By following changes in fluorescence, chemical and thermal stability can be assessed in a truly label-free fashion. The dual-UV technology by NanoTemper allows for rapid fluorescence detection, providing an unmatched scanning speed and data point density. This yields an ultra-high resolution unfolding curves which allow for detection of even minute unfolding signals. Furthermore, since no secondary reporter fluorophores are required as in conventional DSF, protein solutions can be analyzed independent of buffer compositions, and over a concentration range of 150 mg/ml down to 5 µg/ml.Here we demonstrate the performance of the Prometheus NT.48 in monitoring thermal unfolding of various proteins. In screening projects, domain-specific unfolding transition points of antibodies have been quantified, and effects of the conjugation of antibodies with drugs have been assessed. Moreover, stability data from a thermal unfolding detergent screen for integral membrane proteins are presented, demonstrating that label-free thermal unfolding experiments with the Prometheus NT.48 are not affected by amphiphilic and autofluorescent additives. In addition, the precision of the NT.48 is demonstrated in quality control experiments, in which the fraction of unfolded protein in long-time stability or forced degradation tests can be precisely determined by a simple single-scan analysis in a matter of seconds.