Issue link: https://resources.nanotempertech.com/i/1081095
3 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. of up to 48 capillaries at a given temperature takes 3 seconds, and the Prometheus also does not require any calibration times, so that quality control measurements as we describe them here can be performed in under a minute. Results Unfolding of an IgG-antibody as proof-of-concept In order to verify the capability of the Prometheus NT.48 to exactly quantify the fraction of unfolded protein in a given formulation based on the F350/F330 ratio, we created an unfolding standard solution in which IgG antibodies were subjected to controlled thermal unfolding at 80 °C. Unfolded IgG was then mixed with 30 40 50 60 70 80 90 0.75 0.80 0.85 0.90 0.95 1.00 1.05 25 26 27 28 29 30 31 F350/F330 0.80 Temperature (°C) 0 2 4 6 8 10 12 14 16 0.78 0.79 0.8 0.81 0.82 F350/F330 at 25°C % unfolded calculated experimental data IgG thermal unfolding % unfolded 30 40 50 60 70 80 90 0.75 0.80 0.85 0.90 0.95 1.00 1.05 25 26 27 28 29 30 31 F350/F330 0.80 Temperature (°C) 0 2 4 6 8 10 12 14 16 0.78 0.79 0.8 0.81 0.82 F350/F330 at 25°C % unfolded calculated experimental data IgG thermal unfolding % unfolded Figure 2: Establishing a protein unfolding standard. Unfolded IgG at different concentrations was mixed with folded IgG and subjected to thermal unfolding. The percentage of unfolded IgG in the solution was quantified based on the F350/F330 ratio measured at 25 °C. folded IgG to different extends, resulting in formulations with defined amounts of unfolded antibody. In thermal unfolding experiments, all formulations showed similar thermal unfolding profiles with unfolding transition temperatures around 74 °C. However, the initial fluorescence ratio (F350/F330) at 25 °C increased with increasing concentrations of unfolded antibody (Figure 2). A plot of the calculated and experimentally determined percentage of unfolded IgG versus the detected F350/F330 ratio displayed a clear linear correlation as well as a very good agreement between calculated and experimental data. Thus, Prometheus can pick up even small fractions of unfolded IgG (< 0.5 %) in formulations, and could thus be used as a quick and reliable method to follow the stability of antibodies, e.g. during long-term storage tests. Temperature (°C) F350/F330 at 25 °C