47
Melting temperatures are determined using either
intrinsic fluorescence from the protein or using
extrinsic dye. As a protein unfolds, it becomes more
exposed to solution, which changes its fluorescent
properties. This information is used to monitor
unfolding. The unfolding properties of a biologic are
impacted by its environment, just like we see with
DLS-specific parameters.
You can also calculate the T
on
, the temperature at
which unfolding begins. T
on
, also called T
onset
, is, by
definition, always lower than T
m
and is frequently
used to predict aggregation kinetics. A higher T
on
and
a sharper unfolding peak (i.e. when T
on
is close to T
m
)
are considered indicators of higher stability.
There are two additional parameters that are obtained
when DLS is performed during a thermal denaturation
ramp: T
size
and T
scattering
.
T
size
is the temperature at which the average particle
size (r
H
) begins to increase significantly, indicating it
is changing in size from its baseline value. This can be
indicative of domain unfolding, aggregation, and/or
oligomerization. The greater the T
size
value, the more the
formulation resists unfolding and aggregation.
The T
scattering
is the temperature at which the scattering
intensity begins to increase significantly. This usually
indicates large particle formation, i.e. aggregation, and
is also known as T
agg
. These parameters tell you how
your sample behaves under increasing stress, and
are useful parameters for ranking how mutations,
buffers, or conjugates affect your sample's stability.
