-0.002
-0.001
0
0.001
0.002
0.003
0.004
0.005
0.006
Ratio
First
Derivative
0.98
1
1.02
1.04
1.06
1.08
Ratio
350
nm
/
330
nm
30 40 50 60 70 80 90 100 110
Temperature [°C]
T
on
, onset of unfolding
T
m2
, Fab domain
T
m3
, Fc2 domain
T
m1
, Fc1 domain
T
m2
and T
m3
are o en very similar in mAbs,
resulting in only a single transition
(usually labelled T
m2
, but representing
both Fab and Fc2 domain)
T
on
is the point at which unfolding
begins, another useful parameter for
stability assessment
When T
on
and T
m
are close, it
indicates better thermal stability
than when their values differ
This is an example of a typical
unfolding curve of a monoclonal
antibody (NISTmAb, 10.0 mg/ml in
12.5 mM histidine buffer pH 6.0).
The ratio plot (top) shows how
the fluorescence ratio shi s
as hydrophobic residues are
exposed to solution upon thermal
denaturation. The first derivitive
view (bottom) makes it easy to
identify those transitions.
The first T
m
, which corresponds to the
Fc1 domain, is one of the go-to ranking
parameters for mAb candidates
