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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Introduction
Biologics belong to the fastest growing group of drugs,
and an ever growing number of complex molecules
such as mAbs, antibody-drug conjugates (ADCs) and
biosimilars demands novel, predictive analytical
methods to streamline the development process. An
important aspect during drug development is the long-
term stability of the medicinal product [3, 10].
The most critical parameter is the formation of protein
particles, especially at high protein concentrations.
Thereby, protein particles can serve as nucleation seeds
and might promote protein aggregation. Consequently,
particle formation does not only reduce the amount of
active native-like protein, but can also cause unwanted
effects which render the drug ineffective, or even
harmful to patients [2, 9].
In order to predict the aggregation propensity of
biologics, the assessment of their relative thermal
stability and the measurement of ∆G° of unfolding
are becoming invaluable tools during formulation
development and protein engineering [11].
The principle behind the chemical denaturation
approach is the following: By increasing the
concentration of a chaotropic salt, here guanidine
hydrochloride (GuaHCl), protein conformation
equilibrium is shi ed from a folded to a partially
unfolded up to a completely unfolded state. The
fraction of unfolded protein at each measured GuaHCl
concentration can be determined, e.g. by monitoring
changes in the F350/F330 fluorescence ratio. From
this GuaHCl concentration dependence of unfolding,
∆G° can be calculated (Figure 1). Importantly, based
on the Gibbs-Helmholtz law and van´t Hoff equation
(∆G° = - RTlnK), ∆G° can also be used to very precisely
determine the fraction of unfolded protein, which can
then be used to calculate back the fraction of unfolded
protein at [D] = 0 (Table 1).
Figure 1: Chemical denaturation curve of a mAb, analyzed by detecting changes
in the fluorescence emission ratio using the Prometheus NT.Plex. The fit was
performed using a three-state unfolding model.