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4 while the fluorescence was monitored to detect conformational changes by nanoDSF. These experiments showed that the first unfolding transition was indeed reversible at pH 4 (Figure 5A), which is highlighted by a clear refolding signal in the F350/F330 ratio when heating was limited to slightly above T m 1 (60 °C). In contrast, heating beyond the first unfolding transition to 70 °C resulted in irreversible unfolding. At pH 6, the sample was heated to 70 °C (within the first unfolding transition) and 80 °C (beyond the first unfolding transiton) (Figure 5B). In contrast to the experiments at pH 4, no re-folding was observed at pH 6. Albeit the antibody is thermally less stable at pH 4, it is able to re-fold under these conditions to a certain extent, whereas unfolding is irreversible at pH 6. Conclusion The results from our combined investigation of thermal unfolding and aggregation using the Prometheus NT.48 have multiple implications: First and foremost, although pH values > 5.5 thermally stabilize the antibody, collodial stability is compromised, since strong aggregation of the unfolded state occurs. Moreover, even though being thermally less stable at low pH values, unfolding of the antibody is reversible at temperatures up to 60 °C. Taken together, these data suggest that a slight thermal instability might be acceptable and even favourable for long-term stability of the antibody, due to reduced aggregation under these conditions and the ability of the antibody to re-fold. Future screening approaches could therefore be designed to find excipients which thermally stabilize the mAb at lower pH values while maintaining low aggregation of the unfolded state. The presented data demonstrate the versatility and precision of the Prometheus NT.48 which can be used to facilitate formulation development processes. The Prometheus NT.48 detects several stability parameters in an entirely label-free approach. 48 different samples can be analyzed simultaneously, with customizable heating and cooling rates from 0.1 to 7 °C/min. Typical unfolding experiments take just 60 minutes, employing 48 samples, heating rates of 1 °C/min and a temperature ramp from 30 to 90 °C.The on- the-fly detection results in very high datapoint densities (20+ datapoints per minute and capillary) for most precise unfolding analysis. Moreover, sample requirements are low, since only 10 µl of sample with possible protein concentrations between 10 µg/ml and > 250 mg/ml are used per capillary. Figure 4: Summary of T m 1 and T agg values for all tested conditions

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