Chromatographic analysis of drug interactions in the serum proteome

Abstract

The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.

Studies of drug interactions with alpha 1 -acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography

“…These drugs have reported binding constants for AGP that range from 10 4 –10 6 M −1 at or near a physiological pH [6,31]. This range of affinities has been shown in prior work to be suitable for the elution of drugs from columns containing immobilized serum proteins under isocratic conditions and in the presence of a pH 7.4 buffer, such as was employed in this study [1,2]. Table 2 summarizes the normalized retention factors that were obtained for each of these drugs on microcolumns that contained various types of adsorbed AGP, as prepared by using the equivalent of 0.5 mL for serum or an aqueous AGP sample.…”

“…The ability to directly isolate and use such a protein from serum makes this method attractive as a less time-consuming alternative to the covalent immobilization methods that are often used to prepare HPAC columns for drug-protein binding studies [1,2]. In addition, the antibody microcolumns that were developed in this study were sufficiently stable and robust to allow even a single anti-AGP microcolumn to adsorb AGP from various samples and to carry out studies with many drugs or drug concentrations for each application of AGP.…”

“…The use of these methods and tools is not only limited to AGP but could be extended to the study of alternative drug-protein systems by using other antibodies to prepare the microcolumns that are used for on-line immunoextraction. In addition, these methods could be adapted for use in personalized medicine, as demonstrated here in the work with SLE samples, or for examining other types of interactions in biological or clinical samples [2,20]. …”

“…High-performance affinity chromatography (HPAC) is a separation method that has been used in various studies to examine drug-protein binding [1–3]. This technique utilizes a biologically-related ligand as the stationary phase and has been widely applied to the examination of drug-protein interactions by using columns that contain immobilized proteins like AGP or human serum albumin (HSA) [1,2,20]. Advantages of this method include its selectivity, speed, precision, ease of automation, and ability to work with small amounts of a target solute or binding agent [1,2,20].…”

Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes

“…Frontal analysis affinity chromatography has previously been used to determine the binding constants of enzymes [3][4][5][6][7] as well as other affinity interactions including drug-protein binding constants [1,8] and antibody-antigen binding constants [9]. Multiple methods of immobilization exist and include both covalent and noncovalent methods [3].…”