5
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
conditions, and that neither aggregation of the
folded nor unfolded state occur. In order to test
this hypothesis, we compared the chemical
denaturation data with long-term stability
measurements. Here, monomer content and
aggregation were monitored by HPSEC and
turbidity measurements over a time period
of up to 17 months at different temperatures,
respectively. An increase in aggregation over time
results in a decrease in monomer content and an
increase in sample turbidity. Formulation F04 was
identified by both methods to provide best long-
term stability, as it showed the highest monomer
concentrations and lowest turbidity values over
time. This demonstrates that ∆G°
app
-measurements
can robustly predict the long-term aggregation
propensity of mAbs.
In addition to chemical denaturation experiments,
we investigated the conformational and
colloidal stability of the mAb by nanoDSF with
backreflection detection optics. F03 and F04,
which were shown to be most stable in the long-
term turbidity measurements, had the highest
unfolding transition temperatures (75.3 °C and
73.8 °C, respectively) when compared to the
least stable formulations F01 and F02 (69.7 °C
each) (Figure 4). F03 and F04 also had the highest
aggregation onset temperature (86.0 °C and 86.4 °C,
respectively) (Figure 5). Notably, the backreflection
signal intensity scaled directly with the long-term
stability measurements, with F04 showing the
smallest signal at 95 °C (225.2 mAU), while F01 and
F02 showed the highest degree of aggregation
(313.4 mAU and 312.0 mAU, respectively). These
data suggest that the temperature-induced
unfolding and subsequent aggregation detection
using the backreflection optics of the Prometheus
NT.Plex can be used as a predictive method to
evaluate the degree of aggregation propensity of
the unfolded state of biologicals. Thus, quantifying
the degree of aggregation using backreflection
detection could be beneficial when compared
to conventional light-scattering approaches:
Light scattering can typically only be used for T
agg
determination when using concentrated samples,
since the signal is typically lost once strong
aggregation occurs. In contrast, the backreflection
signal quantifies the overall loss in backreflected
light intensity, and can thus qualitatively measure
the overall degree of aggregation even in highly
concentrated samples.
