8
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Conclusion
The presented data demonstrates that chemical
denaturation is a feasible approach to predict the
colloidal long-term stability of biologicals. Moreover,
changes in ∆G° in dependence of the protein
concentration can give immediate feedback whether
protein aggregation occurs, and can also be used to
discriminate between native- and denatured-state
aggregation. Knowledge about the aggregation
mechanism is vital to design further steps in the
development process: Denatured state aggregation
can be reduced by excipients which further stabilize
protein conformation, while native state aggregation
might require different optimization strategies, such
as excipients that shield surface-exposed hydrophobic
patches in aggregation-promoting sequences, or protein
engineering to eliminate such critical sequences entirely
[1, 10].
In summary, we show that the Prometheus NT.Plex is
a uniquely flexible and easy-to-use device that can be
used to extensively characterize the chemical (∆G°),
thermal (T
m
s and T
m
onsets) and colloidal stability (T
agg
and degree of aggregation) of biologicals. The modular
design of the fully automated nanoDSF solution,
comprising the Prometheus NT.Plex in conjunction
with the NT.Robotic Autosampler allows to perform
meaningful chemical denaturation experiments
with sufficient incubation times in-between sample
preparation and measurement.
The Prometheus NT.Plex is a unique and valuable tool
to get the full picture about biologics stability and
aggregation propensity with unprecedented ease-of-
use, flexibility and precision.
Methods
For chemical unfolding experiments, GuaHCl
dilution series were prepared with concentrations
from 6.0 M to 0 M GuaHCl. For this, two stock
solutions with 0 and 6.75 M GuaHCl were prepared
using 5 x formulation buffer which was adjusted
to 1 x with water a er GuaHCl-addition. Arithmetic
dilution series with a final volume of 40 µl were
prepared in 384-well plates (Corning 3820 non-
binding) using a Hamilton Starlet liquid handler.
Preparation time for 16 chemical denaturations
with 24 dilutions each was 1.5 hours. Subsequently,
5 µl of protein were added to each dilution to reach
the desired final concentrations, mixed, sealed
and incubated over night to ensure equilibration
at each concentration. Prepared MTPs were
loaded into the MTP stacker, and capillary chips
