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Review
. 2017 Jun;63(6):1083-1093.
doi: 10.1373/clinchem.2016.262253. Epub 2017 Apr 10.

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

David S Hage  1
Affiliations
Review

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

David S Hage. Clin Chem. 2017 Jun.

Abstract

Background: The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically related binding agent, are 2 methods that can be used to study these interactions.

Content: This review presents various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent.

Summary: The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples.

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Figures

Figure 1
Figure 1
Examples of A) zonal elution and B) frontal analysis experiments for binding studies that were carried out by HPAC. The results given to the right in (A) illustrate the shift in retention that was observed for small injections of R-warfarin (i.e., a drug that binds to Sudlow site I of human serum albumin) onto a column containing immobilized human serum albumin in the presence of various concentrations of the glimepiride in the mobile phase; the vertical dashed line represents the mean position of the peak for R-warfarin in the absence of glimepiride. The results given to the right in (B) show the breakthrough curves that were obtained for the application of various concentrations of carbamazepine to a column that contained immobilized alpha1-acid glycoprotein. Adapted from Refs. (11,13) with permission from Elsevier.
Figure 2
Figure 2
Comparison of data obtained by frontal analysis for examining the binding of R-propranolol and S-propranolol with an HPAC column containing immobilized low density lipoprotein (LDL) (left) and a general model showing the types of interactions each of these enantiomers had with LDL (right). The graph on the left is based on data obtained from Ref. (14).
Figure 3
Figure 3
Use of zonal elution-based competition studies in HPAC to examine the direct competition of a drug or solute with an injected probe for specific binding sites on an immobilized protein, as illustrated by the image on the left for the competition of glimepiride with L-tryptophan at Sudlow site II on columns that contained normal HSA (●) or two glycated preparations of this protein (■,▲). The image on the right shows of modification in glycated HSA and the location of the two major drug binding sites on this protein, Sudlow sites I and II. Reproduced from Refs. (11,24) with permission from Elsevier.
Figure 4
Figure 4
Use of weak affinity chromatography (WAC) to screen the binding of drug fragments, as illustrated by employing a column containing the immobilized ATPase domain of heat shock protein 90 (HSP90). A), chromatograms obtained for fragments 46–62, out of a total of 111 drug fragments tested. B), results obtained for all 111 drug fragments when screened for their binding to HSP90 by using WAC, nuclear magnetic resonance (NMR) spectroscopy, surface plasmon resonance (SPR) spectroscopy, a fluorescence polarization (FP) assay, or a thermal shift assay (Tm), with some results also being included based on X-ray crystallography and isothermal titration calorimetry (ITC). The hits for binding are indicated by green (or dark gray) and non-hits are represented by red (or light gray). Adapted from Ref. (42) with permission from the American Chemical Society.
Figure 5
Figure 5
Scheme for the simultaneous isolation of a free drug fraction and separation of the various chiral forms of a drug in this fraction by using ultrafast affinity extraction and an HPLC chiral stationary phase. This particular example uses an affinity microcolumn containing immobilized HSA for ultrafast affinity extraction. Reproduced from Ref. (73) with permission from The Royal Society of Chemistry.
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