Application Notes

©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 3 APPLICATION NOTE Results Evaluation of two unique families of mAbs IgG-based mAb candidates had mutations introduced in their non-binding regions to profile how single- and double- site mutations altered the stability profiles of these molecules. The aim was to better understand how the genetic and therefore proteomic diversity of early phase antibodies were affected by small changes in the structural core. Each antibody and its mutants were evaluated using nanoDSF, turbidity, and DLS along a thermal ramp to characterize their biophysical characteristics and determine how the mutations affected their stability. When examining the thermal denaturation profiles of the mAbs, it is easy to see their varying behavior. In Figure 1A, four antibodies "Group 1" exhibit different melting temperatures (T m ) via nanoDSF. Additionally, two antibodies (Protein A and Protein D) exhibit significant turbidity accumulation, while Proteins B and C only have modest turbidity signal increase; this is reflected in the cumulant radius data, which also shows an increased cumulant radius at the onset of the experiment for Protein D. For Group 2, the six antibodies show pronounced differences in their nanoDSF unfolding profiles in Figure 1B. Likewise, there are significant differences in their turbidity and cumulant radii changes. Notably, only Protein G shows no major turbidity changes, which is also reflected in its cumulant radius. Detailed parameter measurements can be found in Table 1. Taken together, these results demonstrate the value of simultaneous measurements of DSF, nanoDSF, and backreflection along a thermal gradient. The results demonstrate the structural variation among IgG-derived mAbs, even within related groups. The values for the antibodies can be quantified to evaluate their fitness for development, and to determine how mutations in the genetic sequence can alter biophysical parameters. Nomenclature T m1 First melting inflection temperature, at which 50% of the least thermalstable domain is unfolded T on Onset of unfolding, temperature at which deviation from linear baseline exceeds 0.5% T agg Onset of aggregation, temperature at which scattering deviates >1.1x of initial value (also T size , T scattering ) T turb Onset of turbidity, temperature at which backreflection signal deviates from linear baseline by > 0.5% (Referred to as T agg in Prometheus PR.Plex instruments) PDI Polydispersity Index, a measure of the heterogeneity of particles in a sample; lower values indicate a more homogenous mixture

• Cover