Issue link: https://resources.nanotempertech.com/i/1420877
©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 8 APPLICATION NOTE One of the benefits of adding DLS to the evaluation matrix becomes evident when evaluating T on and T m . In Figure 4A, we show that these parameters are highly, though not perfectly correlated. While characterizing antibodies, when these discrepancies arise, it is important to have additional parameters for evaluation. With DLS, the T scattering and PDI information help resolve cases where the T m and T on do not fall within the expected range. O en these cases indicate unfolded or destabilized species prior to the experiment (via high PDI) or can help validate the rate of unfolding (by monitoring the T scattering ). Likewise, close examination of the size distribution can reveal biophysical changes occurring between the onset and inflection of melting. Figure 5 shows the high correlation between T m1 , T on , T turb (formerly T agg ), and turbidity inflection point (IP) measured by both the Panta and the NT.Plex. It also shows strong correlation with the scattering IP and cumulant radius change IP from DLS data and the thermal unfolding, as measured by the NT.Plex. Notably, measurements related to the DLS parameters of hydrodynamic radius (r H ) and PDI do not have strong correlation to thermal stability behavior. DLS parameters can be used to supplement thermal unfolding-based data to have additional ranking parameters for antibodies. 45 50 55 60 65 70 75 80 PANTA T m1 (ᵒC) PANTA T on ( o C) 75 70 65 60 55 60 55 50 45 40 65 PANTA Tm 1 ( o C) 50 45 55 65 75 85 95 PANTA Scattering IP ( o C) PANTA T on ( o C) 80 70 60 60 55 50 45 40 65 PANTA Scattering IP ( o C) 50 90 Figure 4: Figure 4a demonstrates that while first melting temperature and unfolding onset temperature are correlated, they do not line up perfectly. In 4B, with the additional parameter of scattering IP, we gain deeper insight to the unfolding behavior. Note the antibodies with the higher melting onsets (dark green) cluster along the right side of the graph and can be further refined for beneficial properties using the scattering IP. A B