Characterization of drug–protein interactions in blood using high‐performance affinity chromatography

Abstract

The binding of drugs with proteins in blood, serum or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. Highperformance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug-protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including human serum albumin and α 1 -acid glycoprotein. Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working for directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drugprotein binding will be discussed.

Importance of Relating Efficacy Measures to Unbound Drug Concentrations for Anti-Infective Agents

“…For example, high-performance affinity chromatography (HPAC), a technique coupling high-performance liquid chromatography (HPLC) with affinity columns containing proteins of interest, has been applied to measure the extent of protein binding in blood and characterize the binding process through estimation of equilibrium constants (53)(54)(55)(56)(57). Moreover, immunoaffinity chromatography, a chromatographic method that uses drug binding antibodies, has been applied to measure the free drug fraction of warfarin in a sample containing human serum albumin (58).…”

Plasma protein binding of polyphenols from maritime pine bark extract (USP)

“…The value of each method and the comparability of results is subject of incessant discussions [19][20][21]. For our experimental approach we chose high performance affinity chromatography (HPAC) for fast characterization of the analytes' binding extent to human serum albumin (HSA) [22]. For selected maritime pine bark compounds that were previously detected in human plasma samples [10] we additionally employed a modified ultrafiltration method [23] for comparison of results derived from HPAC.…”

The Significance and Determination of Plasma Protein Binding

“…In both these methods, the protein of interest is used as an immobilized ligand and an injection or application of analyte is made onto the affinity column in the presence of only buffer or buffer plus a mobile phase modifier/competing agent. By examining the elution time or volume of the analyte after it has passed through the column, it is possible to obtain information on the extent of drug-protein binding, the equilibrium constants for these processes, the ability of the drug to compete with other compounds, the effects of temperature or solvent composition on these reactions, and the structure and location of the drug binding sites on the immobilized protein [24].…”