Abstract

High Precision Protein Folding and Aggregation Analytics by NanoDSF

Claire Hatty, Application Specialist, NanoTemper

The assessment of protein stability is of key importance for drug development, drug discovery and basic research. The thermal, colloidal 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 hallmark of the Prometheus NT.48 is the simultaneous label-free, high-throughput fluorimetric analysis of protein folding and the quantification of protein aggregation. Thermal unfolding and aggregation measurements are performed in 48 capillaries in parallel, and require just 10 µl of sample. Since capillary filling does not involve pipetting steps, even highly viscous solutions which are difficult to handle by conventional methods can be easily loaded and analyzed. The unique µl-volume fluorescence detection principle of the Prometheus NT.48 allows for the parallel measurement of samples that can differ 10.000 fold in concentration, with a dynamic protein concentration detection range of a few µg/ml up to > 250 mg/ml. This way, the unfolding transition temperature (Tm) and folding state of proteins can be determined rapidly and with maximal precision - with > 1000 data points per unfolding curve and with measurement errors < 0.1 °C. At the same time, disturbing signals caused by protein aggregation are suppressed, so that exact unfolding transition temperatures can be determined even for strongly aggregating samples. In parallel, temperature-dependent aggregation of proteins can be monitored in a separate channel using backreflection detection, which allows to precisely determine the degree of aggregation as well as the aggregation onset temperature (T agg). Here, we demonstrate the performance of the Prometheus NT.48 in monitoring thermal unfolding of proteins. In screening projects, effects of buffers on thermal and colloidal stability of antibodies have been quantified. 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 Prometheus 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.