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5 Figure 3 Changes in thermal unfolding upon six weeks storage of an IgG1-Antibody in His/Gly- and TRIS buffer: formulations were incubated at 2 °C-8 °C and 40 °C and T m values were measured over a period of 42 days. Here T m values of the F ab fragment are shown. Summary In this Application Note, different applications of the nanoDSF technology as a buffer screening tool have been tested. With the high resolution provided by nanoDSF in thermal unfolding experiments we could resolve unfolding of single antibody domains and showed that the stability of the individual domains is dependent on pH and temperature: the C H 2 domain seems to be most sensitive to lowering pH, in contrast the F ab fragment seems to be most sensitive to temperature stress. These findings show good agreement with already existing literature. The combination of thermal and chemical unfolding studies with short-term stability studies at elevated temperature, show to be a promising tool in finding optimal buffer conditions. In conclusion, the Prometheus NT.48 is well suited for a variety of buffer screening experiments. References 1 http://www.nanotemper- technologies.com/technologies/nanodsf/ 2 Vermeer, A.W.P.; Norde, W.: The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain protein, in: Biophysical Journal, January 2000, Volume 78, Issue 1, pp. 394-404 3 Menzen, T.A.: Temperature-Induced Unfolding, Aggregation, and Interaction of Therapeutic Monoclonal Antibodies (Dissertation), Ludwig- Maximilians-Universität München, 2014, pp. 65-67 4 Garber, E.; Demarest, S.J.: A broad range of Fab stabilities within a host of therapeutic IgGs, in: Biochemical and Biophysical Research Communications, 13 th April 2007, Volume 355, Issue 3, pp. 751-757 5 Thies, M.J.W.: Struktur, Stabilität und Faltung von Antikörperdomänen (Dissertation), Technische Universität München, 2002, pp.93-95, p.135 6 Buchner, J.; et al.: Alternatively folded states of an immunoglobulin, in: Biochemistry, July 1991, Volume 30, pp. 6922-6929 7 Brody, T.: Multistep denaturation and hierarchy of disulfide bond cleavage of a monoclonal antibody in: Analytical Biochemistry, 1997, Volume 247, pp.247-256 8 Brader, M.L.; et al.: Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies, in: Molecular Pharmaceutics, 16 th February 2015, http://pubs.acs.org/doi/abs/10.1021/mp400666b 9 Vermeer, A.W.P.; Norde W.; van Amerongen, A.: The Unfolding/Denaturation of Immunogammaglobulin of Isotype 2b and its F ab and F c Fragments, in: Biophysical Journal, October 2000, Volume 79, Issue 4, pp. 2150-2154