Application Notes

3 destabilization under these conditions. Conversely, the respective buffer substance and salt concentration had little to no effect on the thermal stability of the antibodies. In Figure 3, the thermal unfolding profiles of 3 different antibodies are shown. While antibody B and C show two unfolding events, antibody A shows three distinct unfolding events, which is also evident from the first derivative of the unfolding curves. Conclusion The thermal buffer screen on mAb1 and mAb2 revealed a strong pH-dependence of both mAbs, but little to no effect of the respective buffer substance or the concentration of NaCl. Future screening campaigns could therefore aim to further increase thermal stability at pH-values > 6 by addition of excipients such as glycols or detergents, or by variation of the buffer salts. Our results show that the Prometheus NT.48 delivers highest quality thermal unfolding data for antibody buffer screening campaigns. In summary, 45 different buffer conditions were tested on 2 antibodies, and 90 thermal unfolding curves have been recorded and analyzed within less than 2.5 hours. The large dynamic range of the Prometheus NT.48 allows for analyzing thermal unfolding in solutions containing antibody concentrations between 150 mg/ml down to few µg/ml. Thus, it can be utilized for both, stability screening during early phases of antibody development where only small amounts of protein are available, as well as for final formulation screenings campaigns in highly concentrated samples. The high-resolution of the thermal unfolding curves is also instrumental for biophysical characterization of antibodies. Thus, the effects of buffers, excipients or even covalent modifications on the stability of distinct antibody domains can be easily assessed. Due to the high precision and resolution, nanoDSF poses an attractive alternative to differential scanning calorimetry experiments, which also deliver high resolution unfolding data but require a significant larger amount of protein and time for sample preparation, as well as less throughput and a more limited concentration range. Figure 3 High-resolution data from Prometheus NT.48 thermal unfolding experiments allow for unambiguous discrimination of the unfolding of distinct antibody domains. Plots of the fluorescence ration (F350/F330) and the corresponding first derivative are shown. Material and Methods Protein preparation Antibody stocks (6 mg/ml) were first subjected to a desalting step to remove buffer components from the original formulation, using buffer exchange columns from the NanoTemper Technologies Monolith Protein Labeling kits (www.nanotemper- technologies.com). Antibodies were eluted into H 2 O, and 10 µl of antibody solution were subsequently mixed with 10 µl of 2 x screening buffer in a 384 multi-titer-plate with 25 µl working volume. The antibody solutions were then loaded into nanoDSF grade capillaries (NanoTemper Technologies), and loaded into the Prometheus NT.48 instrument.

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