Application Notes

3 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Results and Discussion Quick analysis of oxidized samples shows differences in unfolding profiles To compare different oxidation levels of a test antibody, recombinant mAb trastuzumab was exposed to 0.3 % H 2 O 2 for 3 and 18 hours, as described previously. 6,7 Samples oxidized for different time periods were analyzed for thermal unfolding using the Tycho NT.6 (Figure 1). Figure 1: H 2 O 2 -triggered oxidation for 3 and 18 hours on trastuzumab results in different folding profiles as measured on the Tycho NT.6 system. Initial ratio is the ratio of the 350 nm/330 nm fluorescence signal detected at the start of the experiment. Inflection temperature (T i ) is the transition point along the curve which represents an unfolding transition of the sample due to thermal treatment. Two inflection temperatures of 74.2 °C and 87.4 °C were detected for the native protein. Tycho NT.6 analysis of the 3- and 18-hour oxidation treatments showed a lower T i 1 for both (68 °C and 66.5 °C respectively) as compared to the native protein. Oxidation has two marked effects on the unfolding profile of trastuzumab. Firstly, the unfolding profile changes significantly with prolonged incubation under oxidative conditions. This is indicated by the shi towards higher values of the initial ratio of detected fluorescence signal. Secondly, a le -shi in the T i values is correlated to the length of oxidation treatment. The shi in T i is larger for the first unfolding event, suggesting that oxidation causes major structural changes particularly in the corresponding domain. In IgG antibodies, the C H 2 domain is usually the first to unfold, followed by the Fab and C H 3 domains as temperature increases. 8 Results from the Tycho NT.6 would suggest that oxidation primarily acts on the C H 2 domain. Binding affinity is influenced by oxidation treatment Since protein A is known to bind IgGs between the C H 2 and C H 3 domains,9 it was used in a binding affinity experiment to confirm that the C H 2 domain of trastuzumab has been affected by oxidation. Binding affinities were identified by using MicroScale Thermophoresis (MST) performed on the Monolith NT.115 Pico system. A dose response curve was generated by titrating the concentration of trastuzumab in the presence of a set concentration of protein A (Figure 2). The resulting K d values show that the higher the oxidation level of trastuzumab, the lower its affinity to protein A. Thus, trastuzumab oxidation, and the resulting destabilization, directly correlated with a decrease in the antibody's ability to bind protein A. A T i value is is the inflection point along the curve which represents an unfolding event due to thermal treatment.

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