Application Notes

3 the fact that antibodies are multi-domain proteins there is more than one inflection point: every domain of an antibody (F ab fragment, C H 2 domain, C H 3 domain) can unfold independently. Temperature or pH value may have different effects on the T m or C m of the individual domains. 2 In the past, DSC- and DSF diagrams of individual fragments and the intact antibody were compared to understand the individual transitions of a multi- domain antibody . 3,4,5 In his DSF analyses, Menzen identified the first melting transition of a monoclonal antibody as the C H 2 domain and the second one as the F ab fragment, whereas the unfolding of the C H 3 domain could not be detected. 3 Thies compared the stability of C H 3 and C H 2: The C H 2 domain proved to be less stable than C H 3 concerning thermal and chemical denaturation. 5 He showed that the large hydrophobic surfaces of the C H 2 domain are strongly exposed to the solvent, which means loss of stability. In contrast, these hydrophobic regions of C H 3 are hidden because of the association of monomeric C H 3 domains, which leads to entropic stabilizations. 5 These assumptions have been used to analyze the unfolding curves provided by the nanoDSF technology of the Prometheus NT.48. The denaturation curves show that the IgG1- Antibody produces two peaks deriving from at least two domains, which denature under distinct conditions (Figure 1). The first transition point correlates with the C H 2 domain and the second one with the F ab fragment. A transition point of the C H 3 domain could either not be detected by the Prometheus NT.48 or may be overlaid by the F ab peak, since the latter peak seems to be quite broad. Figure 1 Formulation screening of an IgG1-Antibody as a tool to find optimal conditions for increased protein stability (A) Chemical unfolding curves in His/Gly buffer at different pH values at 20 °C (B) Corresponding fir st derivative indicating the denaturation midpoints C m 1 and C m 2 (C) Thermal unfolding curves in TRIS buffer at different pH values (D) Corresponding first derivative indicating transition midpoints T m 1 and T m 2

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