Measurement of Drug−Protein Dissociation Rates by High-Performance Affinity Chromatography and Peak Profiling

Abstract

The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates.

Analytical methods for kinetic studies of biological interactions: A review

“…The retention and separation of a target analyte from other sample components by this method is based on the specific and reversible interactions that occur in many biological interactions [6,8,9,107–111]. High-performance affinity chromatography (HPAC) is a type of affinity chromatography that uses the supports and instrumentation of high-performance liquid chromatography to provide both rapid and efficient separations based on biological interactions and affinity ligands [112]. The development of HPAC has also lead to the creation of various new or improved techniques that can use affinity chromatography to study the kinetics and thermodynamics of biological interactions [6,112,113].…”

“…Peak profiling is a related method that examines the band broadening of a target and a non-retained solute on an affinity column (and possibly on a control column) under linear elution conditions [106,112]. Eq.…”

“…Eq. (19) is often used in peak profiling studies to provide information on the dissociation rate constant k −1 from band broadening data [112,118–120]. …”

“…Eq. (19) can be used with data obtained at a single flow rate to calculate the value of k −1 , or data acquired at several flow rates can be used to construct a plot of ( H R − H M ) versus ( u k )/(1 + k ) 2 and the dissociation rate constant can be obtained from the slope of the best-fit response [112,118–120]. …”

Biological assessment of neonicotinoids imidacloprid and its major metabolites for potentially human health using globular proteins as a model

“…However, sparse systematic work has been done on the binding of neonicotinoids with several classically proteins. Investigations into the more delicate effects of the binding phenomena between neonicotinoids and proteins can supply significant perception for the general toxicity of these agrochemicals, as the protein-ligand of a compound in this manner may adjust its allocation in the body, and thereby influence both the dose-response connection and the speed of compound elimination, and ultimately the toxicological responses in the body [25][26][27][28][29]. The present investigation was schemed to delve the binding of imidacloprid, which is regarded as one of the typical neonicotinoids, with two representatively mammalian proteins, i.e.…”