Application Notes

Getting the full picture: predicting the aggregation propensity of mAbs using chemical and thermal denaturation on a single, fully automated platform

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3 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Different aggregation mechanisms have several effects on protein stability, and thus on ∆G°. Since aggregation depends on the overall protein concentration, additional information about the aggregation mechanism can be extracted by measuring ∆G° at different protein concentrations. There are three possible outcomes of this approach [8]: i) ∆G° is independent of protein concentration, which means that there are no intermolecular interactions and no aggregation propensity. ii) ∆G° increases with increasing protein concentrations, which means that the folded state of the protein is stabilized by "native-state" aggregation. And iii) ∆G° decreases with increasing protein concentration, which means that the unfolded state becomes more populated by irreversible "denatured-state" aggregation. The latter can also be accelerated using thermal unfolding in temperature ramps, which can then be detected with the Prometheus NT.Plex with aggregation detection optics, thereby providing direct feedback on conformational and colloidal stability of proteins. In this study we performed proof-of-concept chemical denaturation experiments with lysozyme, which is very well characterized in terms of aggregation pathways. Moreover, we analyzed ∆G° app , aggregation onset temperatures (T agg ) and unfolding transition temperatures (T m ) of a mAb in different formulations, and compared the results with turbidity and monomer content over time as assessed by HPSEC for long- term stability data. For this, we used automated liquid handling in conjunction with capillary-chip filling using the NT.Robotic Autosampler and automated measurement execution by the Prometheus NT.Plex. The results show that this combinatory approach of thermal and chemical denaturation allowed for the identification of the formulation with the best long-term stability. Table 1: Calculated values illustrating the correlation between ∆G° and the fraction of denatured protein at [D] = 0.

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