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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Results
In order to test the capability of chemical
denaturation to predict protein aggregation, we
first investigated lysozyme unfolding in presence
of GuaHCl at different buffer pH values. Lysozyme
is known to undergo irreversible, denatured-
state formation of fibrils and aggregates at pH
values ≥ 12.2 [4, 5, 7], whereas it is stable at low
salt condition at pH 5.2. Therefore, we measured
changes in ∆G°
app
at these two pH values at different
lysozyme concentrations. Figure 2 shows a strong
lysozyme-concentration dependent decrease in
∆G°
app
in phosphate buffer pH 12.2, whereas ∆G°
app
is independent of lysozyme concentration in
70
60
50
40
30
20
10
0
0 50 100 150 200 250 300 350 0
DG
(kJ/mol)
Acetate pH5.2
Phosphate pH 12.2
Lysozyme (µM)
acetate buffer pH 5.2. These results indicate that
chemical denaturation is suited to identify buffer
conditions which promote protein aggregation,
and are in good agreement with studies using the
osmotic second virial coefficient to predict colloidal
stability [6].
Next, we investigated a monoclonal antibody,
mAb1, in 5 different formulations. Preparation
of dilution series, filling of capillary chips and
assay execution were fully automated using the
Prometheus NT.Plex in conjunction with a liquid
handling system and the NT.Robotic Autosampler.
For each condition, 24 GuaHCl concentrations
between 0 and 6M at mAb concentrations of
20 mg /mL, 5 mg/mL, 2 mg/mL and 0.5 mg/mL,
respectively, were prepared and incubated over
night to ensure sample equilibration.
In 4 out of 5 formulations a marked decrease
of ∆G°
app
could be observed at increasing mAb
concentrations, suggesting that the denatured
state of the mAb has a strong tendency to form
irreversible aggregates (Figure 3). Only formulation
F04 showed no change in ∆G°
app
, suggesting that
the colloidal stability is maximal under these
Figure 2: Chemical denaturation of lysozyme at different pH values. The
decrease in ∆G°
app
at pH 12.2 indicates denatured-state aggregation. Lines are
guidance to the eye.
