5
Figure 5: Testing the reversibility of the first unfolding transition event of a mAb at (A) pH 4 and (B) pH 6. mAb re-folding can be
observed after incomplete unfolding in acetate buffer pH 4 (top left).
Methods
Sample preparation:
The purified mAb (49.8 mg/ml in PBS pH 6.2) was
diluted into buffer to reach a final concentration of
5 mg/ml mAb, 20 mM acetate at indicated pH
values, and optionally 130 mM NaCl. Prior to
thermal stability experiments, the solutions were
incubated for 1 h at RT.
Thermal unfolding and aggregation
experiments:
20 µl of each sample were loaded into 2
Prometheus NT.48 standard treated capillaries.
Capillaries were placed into the instrument and
thermal unfolding and aggregation was monitored
over a temperature range from 40 to 90 °C, with a
heating rate of 1 °C/min. Fluorescence at 330 and
350 nm as well as light extinction were detected in
parallel. For refolding experiments, 20 µl of each
sample were loaded into 2 Prometheus NT.48
standard treated capillaries. Capillaries were
placed into the instrument and thermal unfolding
and aggregation was monitored over a temperature
range from 20 to 60, 70 or 80 °C, respectively, with
a heating rate of 1 °C/min, and subsequently
cooled down to 20 °C at 1 °C/min.
