Application Notes

Thermal stability buffer screening of therapeutic antibodies

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2 screen addressed the thermal unfolding transition temperatures of the antibodies in dependence of pH, salt and buffer substances. The buffer screen comprised the buffer substances Na-Citrate (pH 3.5-6), Na-Acetate (pH 3.2-6), NaPO 4 (pH 6-8.5), and Tris (pH 7-8.5), each at 25 mM final concentration, each in the absence and presence of 130 mM NaCl. Thermal unfolding experiments were carried out on the Prometheus NT.48 at a heating rate of 1 °C/min. Both antibodies showed multiple unfolding transitions in the plot of the fluorescence ratio (F330/F350) versus temperature (Figure 2A and B). The distinct unfolding events can be attributed to the different thermal stabilities of FAB- and FC-domains of the antibody. Figure 2 Thermal unfolding curves and unfolding transition midpoints of mAb1 and mAb2. (A) Thermal unfolding curves of mAb1 and mAb2 in presence of 25 mM Na-Citrate at different pH values. Insets show the pH-dependence of the first unfolding transition midpoint (Tm1). (B) Dependence of Tm1 and Tm2 on the pH of the buffer for all tested conditions. A plot of the unfolding transition temperature versus pH revealed that the thermal stability of both mAbs strongly depended on the pH of the buffer: While both antibodies displayed constant thermal stabilities at a pH range from 6 to 8.5, with Tm1- values around 70 °C, a strong decrease in unfolding transition temperatures at pH values < 6 was observed, pointing towards a significant

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