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
