Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Figure 2: Oxidation treatment of trastuzumab influences its affinity for binding to protein A. Dose-response curves for the interaction of trastuzumab with fluorescently labeled protein A was generated using MicroScale Thermophoresis (MST) run on the Monolith NT.115 Pico system. Error bars indicate standard deviation from triplicate measurements. The dissociation constant (K d ) is a measure of the concentration of a ligand where the binding site on a particular protein is half occupied. Oxidation of the mAb resulted in reduced K d values as compared to the native sample. In summary, trastuzumab's unfolding profile is influenced by oxidation treatment, which also translates into reduced binding capabilities (Table 1). Table 1: Summary of key parameters from Tycho NT.6 experiment. Following up this analysis using the Monolith NT.115 Pico system provided detailed confirmation of the influences of oxidation on antibody interactions. Having the tools to rapidly and accurately monitor and discern the unfolding characteristics of an antibody when subjected to external challenges such as oxidation, will enable researchers to gain a better understanding of the functionality of their targets and may ultimately accelerate development of better therapeutics. Materials and Methods Forced antibody oxidation The recombinant monoclonal antibody trastuzumab was diluted with phosphate buffered saline (PBS) to 15 mg /mL. To trigger artificial oxidation, the sample contained hydrogen peroxide (H 2 O 2 ) at a final concentration of 0.3% and incubated at room temperature in the dark for 3 and 18 hours. The native antibody sample contained 15 mg /mL of antibody in PBS without addition of H 2 O 2 and was also incubated at room temperature in the dark for 3 and 18 hours. The reaction was terminated by buffer exchange with cooled (4 °C) PBS supplemented with 0.05% Using the Tycho NT.6 to rapidly monitor the unfolding profile of an antibody (or any kind of protein) can provide information on the functionality of the target protein and can also be used to identify critical steps in the downstream processing workflow.

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