4
Thermal and chemical unfolding
Thermal unfolding experiments rely on forced
degradation assays, where biotherapeutics
are subjected to increasingly high temperature
causing them to unfold. This process results in
a prediction of the melting temperature (T
m
) at
which 50% of the mAb population is unfolded
1
.
Chemical unfolding experiments work in a similar
way, but use chemical denaturants such as
guanidine hydrochloride or urea to fully denature
proteins. From the unfolding curves, researchers
can calculate the change in the Gibbs free
energy of unfolding, ΔG, and the inflection point,
C
1/2
. Theoretically, the greater the ΔG, C
1/2
, or T
m
, the
more structurally or conformationally stable the
molecule
2
. Next are the most utilized techniques
to measure thermal and chemical unfolding of
biotherapeutics.
Differential scanning calorimetry (DSC) a
microcalorimetry thermal unfolding technique
that measures heat capacity as a function of
temperature; it predicts thermal stability. Usually,
researchers place a mAb solution into a fixed
sample cell and a corresponding buffer into a
reference cell. They then compare the heat capacity
(Cp) signal from the sample cell to the reference
cell. As temperature increases, the temperature
difference between reference and sample cells is
continuously measured and calibrated to power
units. The technique is considered the standard for
testing unfolding and stability, but requires large
sample volume.
Differential scanning fluorimetry (DSF)
measures mAb thermostability through
