A mAb was subjected to an incremental cycling experiment. The initial temperature was
45 °C, and each cycle increased temperature by 4 °C with respect to the previous cycle. The
overlaid thermal unfolding curves demonstrate that profound differences in the unfolding
pattern can be observed between concentrated (blue, 10 mg/ml) and diluted (red, 40 µg/ml)
mAb solutions. While the dilute solutions clearly show reversibility after unfolding at 61 °C,
mAb concentrations in the mg/ml range display irreversible unfolding. The identical test was
performed with a second mAb
(shown below), where no such
effect of protein concentration
on unfolding reversibility was
observed. These data
demonstrate that this is a
protein-specific property. In
conclusion, dilution effects can
differentially affect proteins and
can alter the outcome of
biophysical characterizations of
mAbs, so that analysis should
be preferably performed at
concentrations which mimic the
final product.
Conclusion
The presented data and applications highlight the versatility of the Prometheus instrument for
the characterization of proteins. The new PR.TimeControl software enables straightforward
evaluation of unfolding reversibility and kinetics with an unprecedented ease-of-use,
precision and simplicity. The experiments shown here also demonstrate a wealth of
information that has not been easily accessible so far and which will help to streamline
workflows and enable informed decisions already in early stage of development. The
analysis of protein folding reversibility using the Prometheus series has therefore the
potential to become an essential part of the biophysical characterization in biopharmaceutical
development and enzyme engineering.
