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Review
. 2012 Oct:69:93-105.
doi: 10.1016/j.jpba.2012.01.004. Epub 2012 Jan 14.

Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments

David S Hage  1 , Jeanethe A Anguizola, Cong Bi, Rong Li, Ryan Matsuda, Efthimia Papastavros, Erika Pfaunmiller, John Vargas, Xiwei Zheng
Affiliations
Review

Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments

David S Hage et al. J Pharm Biomed Anal. 2012 Oct.

Abstract

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered.

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Figures

Figure 1
Figure 1
Separation of R- and S-warfarin on a 10 cm×4.6 mm I.D. silica monolith column containing immobilized α1-acid glycoprotein. These separations were obtained at room temperature using pH 7.0, 0.067 M phosphate buffer as the mobile phase. The inset compares the total plate height (Htotal) that was measured for S-warfarin on the AGP silica monolith column to the total plate height that was determined for the same target and affinity ligand when using 7 ìm silica particles as the support. Adapted with permission from Ref. [17].
Figure 2
Figure 2
The on/off elution format for affinity chromatography and a general chromatogram for this format.
Figure 3
Figure 3
(a) Injection of R-warfarin onto an inert control column and (b) affinity extraction of R-warfarin by an immunoaffinity microcolumn of the same size but that contained anti-warfarin antibodies. The contact time between the injected sample and the immunoaffinity layer in (b) was only 60 ms. Adapted with permission from Ref. [38].
Figure 4
Figure 4
Protocol used with multiple lectin affinity chromatography for the study of glycoproteins in serum. Four techniques were used in this separation: immunodepletion, glycoprotein fractionation, isoelectric focusing (IEF) fractionation using a digital proteome chip (dPC), and reversed-phase liquid chromatography in conjunction with mass spectrometry (LC-MS). Reproduced with permission from Ref. [55].
Figure 5
Figure 5
Synthesis of a monolith containing a boronate, as based on in situ free radical polymerization using 4-(3-butenylsulfonyl) phenylboronic acid and N,N-methylenebisacrylamide. The azobisisobutyronitrile (AIBN) was used to initiate polymerization. The porogen was a mixture of two solvents that was used to promote the formation of pores in the monolith during its formation. Adapted with permission from Ref. [73].
Figure 6
Figure 6
An example of a displacement immunoassay. Adapted with permission from Ref. [40].
Figure 7
Figure 7
Scheme for the purification and mass spectrometric analysis of a target by using surface-enhanced laser desorption/ionization (SELDI) with a surface that contains immobilized metal ions. Based on information provided in Ref. [99].
Figure 8
Figure 8
Examples of (a) a zonal elution competition study with R-warfarin and gliclazide as a competing agent on an HSA column and (b) a frontal analysis experiment for gliclazide applied to an HSA column, with data being fitted to a two-site binding model. Terms: k, retention factor; mL,app, moles of applied target that are needed to reach the mean position of the breakthrough curve at a given concentration of the target. Adapted with permission from Ref. [121].
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