Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. differences between the determined unfolding transition temperatures between the methods may be attributed to the different detection methods. In the context of formulation screening it is important to see that the relative correlations between the different formulations are virtually identical. Consequently, stability screenings based on T m results in the same ranking by nanoDSF and µDSC. Importantly, nanoDSF requires significantly less sample and time for formulation screening projects when compared to µDSC (Figure 2B) while delivering highly comparable T m -values. Conclusion In this comparative study we demonstrate that both methods, nanoDSF and µDSC, provide precise and consistent data. However, µDSC has several limitations, as listed in Figure 2C. nanoDSF integrated in the Prometheus NT.48 overcomes these drawbacks by its innovative capillary format. It allows for easy sample handling, even for highly concentrated and very viscous formulation conditions, and for providing a maintenance-free instrumentation which does not require laborious instrument equilibration and washing. In addition to its speed, precision and throughput, nanoDSF is a particularly robust method which does not request any cumbersome sample preparation such as dialysis or filtration (Figure 2C) and also works in any buffer, even with detergents and high viscosity (> 50 mPa). Therefore, the Prometheus NT.48 is the ideal instrument for rapid and precise thermal stability screening in biopharmaceutical development. µDSC detects unfolding transitions in temperature gradients as distinct "peaks" in the heat capacity of the solution in respect to a reference measurement. In contrast, nanoDSF detects unfolding events by recording changes in the emission properties of the environment-sensitive amino acids tryptophan and tyrosine. Typically, exposure of tryptophan residues from the hydrophobic protein core to the aqueous formulation results in a shi of the emission maximum to higher wavelengths, and therefore to an increase in the F350/F330 ratio. Both methods, nanoDSF and µDSC are very sensitive and allow for detecting multiple unfolding events. mAbs, for example, show multiple unfolding events, which can be attributed to their different domains. In the present case, most likely, the first unfolding transition (T m 1) corresponds to unfolding of the CH2 domain, while the second transition (T m 2) reflects simultaneous unfolding of the CH3 domain and Fab. 1 Comparison of T m data from nanoDSF and µDSC data show a good agreement between the methods (Figure 2A). Importantly, the standard deviation of triplicate T m determination by nanoDSF was on average 0.1 °C for all measurements, highlighting the high precision of the method. Both methods find that the mAb is least stable in acetate buffer pH 3, while its stability substantially increases with increasing pH. µDSC and nanoDSF both confirm that the mAb is thermally most stable in histidine buffer at pH 7, and that the surfactants PS20 and PS80 do not have a positive effect on thermal stability, but instead lower the T m 1 value by ~2.2 °C. Slight

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