Application Notes

Fast and accurate evaluation of oxidation-induced destabilization of mAbs

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APPLICATION NOTE Tycho is a trademark of NanoTemper Technologies GmbH, Munich, Germany. NanoTemper is a registered trademark of NanoTemper Technologies GmbH, Munich, Germany. ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. NT-TY-001-01 Tween-20. Up to three buffer exchange steps were carried out with protein desalting columns to remove excess hydrogen peroxide. Tycho NT.6 experiments The recovered antibody samples, a er 3 and 18 hour incubations with hydrogen peroxide, were diluted with PBS supplemented with 0.05% Tween-20 to a concentration of 1 mg /mL for Tycho NT.6 experiments. Samples were loaded as duplicates into Tycho NT.6 Capillaries (NanoTemper Technologies, Cat# TY-C001) and thermal unfolding profiles of native and oxidized trastuzumab were recorded. Temperature inflection values (T i ) were obtained by automated data analysis. Monolith NT.115Pico experiments 1 mg /mL of protein A (Thermo Fisher Scientific) was labeled with the fluorescent dye RED-NHS 2nd Generation according to the the Monolith NT Protein Labeling Kit RED–NHS protocol (NanoTemper Technologies, Cat# MO-L001). Subsequently, interaction of labeled protein A to native and oxidized trastuzumab was measured in PBS supplemented with 0.05% Tween-20. The concentration of labeled protein A was constant at 5 nM, and trastuzumab was added in a serial dilution at final concentrations between 500 nM and 30 pM. Samples were loaded into Monolith NT.115 Premium Capillaries (NanoTemper Technologies, Cat# MO-K025) and measured on a Monolith NT.115 Pico system using 5% excitation power and high MST power to determine K d values. Data was analyzed using MO.Affinity Analysis so ware version 2.2.4. References 1. Lagassé, H. A. D. et al. Recent advances in (therapeutic protein) drug development. F1000Research 6, 113 (2017). 2. Brekke, O. H. & Sandlie, I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat. Rev. Drug Discov. 2, 52–62 (2003). 3. Wang, W., Singh, S., Zeng, D. L., King, K. & Nema, S. Antibody structure, instability, and formulation. J. Pharm. Sci. 96, 1–26 (2007). 4. Vlasak, J. & Ionescu, R. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Curr. Pharm. Biotechnol. 7, 92426–2447 (2008). 5. Bertolotti-Ciarlet, A. et al. Impact of methionine oxidation on the binding of human IgG1 to FcRn and Fc receptors. Mol. Immunol. 46, 1878–1882 (2009). 6. Loew, C. et al. Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies. J. Pharm. Sci. 101, 4248–4257 (2012). 7. Wang, W. et al. Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. Mol. Immunol. 48, 860–866 (2011). 8. Voynov, Vl et al. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation. PLoS ONE 4(12): e8425 (2009) 9. Deisenhofer, J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-Å resolution. Biochemistry 20(9), 2361–2370 (1981)

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