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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Different aggregation mechanisms have several effects
on protein stability, and thus on ∆G°. Since aggregation
depends on the overall protein concentration,
additional information about the aggregation
mechanism can be extracted by measuring ∆G° at
different protein concentrations. There are three
possible outcomes of this approach [8]:
i) ∆G° is independent of protein concentration,
which means that there are no intermolecular
interactions and no aggregation propensity.
ii) ∆G° increases with increasing protein
concentrations, which means that the folded
state of the protein is stabilized by "native-state"
aggregation. And
iii) ∆G° decreases with increasing protein
concentration, which means that the unfolded
state becomes more populated by irreversible
"denatured-state" aggregation. The latter can
also be accelerated using thermal unfolding in
temperature ramps, which can then be detected
with the Prometheus NT.Plex with aggregation
detection optics, thereby providing direct
feedback on conformational and colloidal
stability of proteins.
In this study we performed proof-of-concept chemical
denaturation experiments with lysozyme, which
is very well characterized in terms of aggregation
pathways. Moreover, we analyzed ∆G°
app
, aggregation
onset temperatures (T
agg
) and unfolding transition
temperatures (T
m
) of a mAb in different formulations,
and compared the results with turbidity and monomer
content over time as assessed by HPSEC for long-
term stability data. For this, we used automated liquid
handling in conjunction with capillary-chip filling
using the NT.Robotic Autosampler and automated
measurement execution by the Prometheus NT.Plex.
The results show that this combinatory approach of
thermal and chemical denaturation allowed for the
identification of the formulation with the best long-term
stability.
Table 1: Calculated values illustrating the correlation between ∆G° and the
fraction of denatured protein at [D] = 0.