Technology Brief 3
nanoDSF
Evaluation of slow equilibration kinetics in antibody chemical
denaturation experiments
With the Prometheus NT.Plex
Chemical denaturation is becoming increasingly popular in the drug development process. On one
hand, it allows for determining the free enthalpy (ΔG) of unfolding of monoclonal antibodies
(mAbs), which directly correlates with the fraction of unfolded protein in solution and provides
thermodynamic insight into mAb stability. On the other hand, it is a tool to predict the aggregation
propensity and aggregation mechanism of mAbs. Chemical denaturation experiments are
performed by measuring the fraction of unfolded protein (e.g. by measuring the F350/F330 ratio) in
a gradient of denaturant concentrations. Common denaturants are 0-8 M GuaHCl, 0-10 M Urea. In
order to record a sufficient number of data points for fitting, at least 24 different denaturant
concentrations should be measured. In order to derive correct ΔG values from chemical
denaturation experiments, it is of major importance to use fully equilibrated samples. The time
required for full equilibration is however often unknown, and some of the current commercial
instrument solutions to detect chemical unfolding combine sample preparation and detection,
which might lead to unsufficient incubation times.
Here we used the Prometheus NT.Plex to measure the isothermal equilibration of chemical
denaturation reactions for three different mAbs at GuaHCl concentrations between 0 and 7 M at
20 °C:
The results show that large changes in the F350/F330 ratio can be observed over time at GuaHCl
concentrations at which the mAb is only partially unfolded. Under these conditions, even an
incubation time of 10 h was not sufficient to reach full equilibration of all samples.
