As illustrated in Figure 3, fluorescence data is collected at a series of denaturant concentrations, and the raw data is fit to a protein unfolding transition state model. The calculated thermodynamic parameters ΔGº, m, and C1/2 are determined by nonlinear least-squares fitting of the data to the model. ΔGº is the Gibbs free energy of protein unfolding in the selected buffer, m is the rate of change in ΔG as a function of denaturant concentration, and C1/2 is the denaturant concentration at which one-half the protein molecules are in the native state and one-half are in the unfolded state. Typically, ΔGº and C1/2 increase as the stability of a biologic is improved. Protein constructs, formulations, and process conditions can all be rank-ordered for stability using the ΔGº and C1/2 parameters.
Chemical denaturation can also be measured with other physical observables like ultraviolet spectroscopy, dynamic light scattering, or circular dichroism. In addition to ΔGº, m, and C1/2 values, these different measurements can provide specific structural information.
For example, while tryptophan fluorescence provides information about the solvent exposure of those residues, circular dichroism reports changes in secondary structure. Different endpoint values may be used to assess the degree of unfolding or the environment of the fluorophores upon denaturation. In the case of tryptophan fluorescence, this additional information is contained in the emission spectrum.
Fluorescence measurements can also be performed with extrinsic fluorescence probes (such as ANS, Sypro Orange, Nile Red, and Thioflavin T), which have the added advantage of being sensitive to additional processes such as aggregation or the formation of protein fibers. Whether using either intrinsic or extrinsic fluorescence, the new instrument completely automates the chemical denaturation experiment. When used in conjunction with other instruments (such as dynamic light scattering or circular dichroism), the 2304 can be used for automated sample processing for subsequent off-line analysis.
In addition to rank-ordering by stability, biologics can be rank-ordered by propensity to aggregate. This application is also accomplished automatically by the AVIA instrument. In this case, the denaturant (typically urea or guanidinium chloride) is substituted with an ammonium sulfate solution. By monitoring 90º light scattering (using the same detector) as a function of ammonium sulfate concentration, different protein constructs or different formulations can be rank-ordered directly by propensity to aggregate.