Evaluation of slow equilibration kinetics in antibody chemical denaturation experiments

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.

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