Application Notes

Comparison of nanoDSF and µDSC for thermal stability assessment during biopharmaceutical formulation development

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2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. To directly compare the precision and overlap of the T m -determination by µDSC and nanoDSF, we conducted a small formulation screen using a commercial, therapeutic mAb. Results We analyzed thermal unfolding of the mAb in a total of ten diff erent formulations with varying buff ers and pH-values. We also analyzed thermal unfolding in changes in the heat capacity (C p ) of a protein- containing solution relative to a reference solution. µDSC can be used to calculate unfolding transition temperatures as well as thermodynamic stability parameters. Although typically considered the gold standard for thermal stability measurements, µDSC has several drawbacks. Due to technical reasons rigorous equilibration and calibration is required, which preclude a parallelization of measurements so that samples have to be measured one-at-a-time. Moreover, the concentration range is limited to about 0.5 to 5 mg/ml mAb. Sample volumes of typically hundreds of µl per sample are required, which sum up quickly in screening campaigns. Thus, a pre-selection of conditions is o en performed to minimize sample consumption. In order to optimize the screening procedure for the identifi cation of ideal formulation conditions, a higher throughput and lower sample consumption are desired while maintaining precise T m detection. The Prometheus NT.48 instrument fi lls this gap by analyzing protein unfolding transitions based on high- precision detection of intrinsic fl uorescence changes. This truly label-free approach allows for the parallelized detection of up to 48 samples with concentrations ranging from 10 µg/ml to more than 250 mg/ml without buff er restrictions. It does not require the addition of extrinsic fl uorescent dyes like in the classical DSF technique, avoiding potential detrimental interactions of dye and protein or excipients. The innovative dual-UV detection method enables rapid scanning of samples, which results in a very high datapoint density of 20 or more datapoints per °C depending on the steepness of the temperature ramp. Figure 1: Thermal unfolding data for a mAb at a concentration of 1 mg/ml in two diff erent buff er conditions, recorded by nanoDSF (red) and µDSC (blue). nanoDSF T m values are determined from the transition midpoints (corresponding to peaks in the fi rst derivative of the F350/F330 data, red dotted line). µDSC T m values are determined from peaks in the heat capacity C p .

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