Theoretical Studies of Solute-Binding Agent Interactions in Ultrafast Affinity Extraction

Ultrafast affinity extraction (UAE) is a method by which the association equilibrium constant (Ka) and dissociation rate constant (kd) for a drug or solute with a binding agent can be determined in solution, giving properties that are useful for describing solute and binding agent interactions. Three fitting methods - linear, exponentially-modified Gaussian (EMG), and quadratic - were compared to determine which gave the best fit to chromatographic peaks that were obtained in UAE systems. The results showed that solutes with larger equilibrium constants with their binding agents gave a better fit of their UAE peaks when using linear or EMG functions, while solutes with low binding affinities gave a better fit when using the quadratic function. The theoretical response of UAE was also examined by using equations derived from chromatographic theory to look at how varying the measured free fraction of a solute and the solute-to-binding agent ratio altered the apparent equilibrium constants that were estimated by UAE. The effects of altering the dissociation rate constant for the interaction and original free fraction on the apparent measured free fraction were also modelled. These data were created to better understand the conditions under which UAE should be performed to provide accurate estimates of equilibrium constants and rate constants. The theoretical results generated were consistent with experimental data but also indicated that some further refinements were needed in the models that were used in these studies