Application Notes

Fast and accurate evaluation of oxidation-induced destabilization of mAbs

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2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Trastuzumab, also known as the commercial drug Herceptin, is a therapeutic monoclonal antibody that has been used to successfully treat patients with certain forms of breast cancer for almost 20 years. Specifically, trastuzumab acts by binding to and interfering with the HER2/neu receptor in cancer patients. Having the ability to quickly monitor relative stability and functionality of a protein will result in better antibody development. In this study, we evaluate the effects of oxidative stress on the thermal unfolding profile of the humanized monoclonal antibody IgG1 trastuzumab using the Tycho NT.6 system. We show that trends in the unfolding profile quickly determined by the Tycho NT.6 correlate with the antibody's capability to bind to protein A, as seen using the Monolith NT.115 Pico system. The results provide insight into the effectiveness of this mAb to act as a therapeutic binding protein once degradation due to improper storage and handling has occurred. A fast methodology to evaluate degradation and assess antibody functionality as a consequence of oxidation effects would enable protein production researchers to develop better antibody-based applications. rejection. 2 For protein production researchers working on these antibody-based therapeutic drugs, optimizing manufacturing and storage conditions is a critical step in their process development. Some of these degradation processes lead to the loss of biological activity or efficacy and even potentially cause adverse immunogenic reactions when administered in patients. 3 Similarly, antibodies used in basic research as well as diagnostics studies can be rendered unreliable due to degradative processes. It has been shown that degradation at the antigen binding site as well as in the Fc region impact binding affinity towards the antigen and the FC-receptor, and therefore influence antibody function. 3,4 One step of the chemical degradation pathway is the oxidation of methionine (Met) residues in the constant region of antibodies. Met residues are readily oxidized, and it has been reported that Met oxidation affects the antibody's affinity or interaction with the neonatal Fc receptor and Fc receptor, 5 as well as the binding to protein A 6 , by destabilizing the antibody. All proteins, including antibodies, are susceptible to chemical and physical destabilization processes if improperly handled or stored.

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