High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents

Abstract

The last few decades have witnessed the development of many high-performance separation methods that use biologically related binding agents. The combination of HPLC with these binding agents results in a technique known as high performance affinity chromatography (HPAC). This review will discuss the general principles of HPAC and related techniques, with an emphasis on their use for the analysis of biological compounds and pharmaceutical agents. Various types of binding agents for these methods will be considered, including antibodies, immunoglobulin-binding proteins, aptamers, enzymes, lectins, transport proteins, lipids, and carbohydrates. Formats that will be discussed for these methods range from the direct detection of an analyte to indirect detection based on chromatographic immunoassays, as well as schemes based on analyte extraction or depletion, post-column detection, and multi-column systems. The use of biological agents in HPLC for chiral separations will also be considered, along with the use of HPAC as a tool to screen or study biological interactions. Various examples will be presented to illustrate these approaches and their applications in fields such as biochemistry, clinical chemistry, and pharmaceutical research.

Figure 1

Steps involved in the on/off elution mode of affinity chromatography. The analyte, or target, that can be retained by the immobilized binding agent is represented by the circles, while non-retained sample components are represented by the diamonds.

Figure 2

General schemes for (a) a chromatographic displacement immunoassay and (b) a chromatographic sandwich immunoassay.

Figure 3

Chromatogram obtained by combining protein A and size-exclusion HPLC columns for the analysis of monoclonal antibodies versus other components in a Chinese hamster ovary (CHO) cell culture. The peaks for the monoclonal antibodies and their aggregates are shown to the right, after their elution from the protein A column and loading onto the size-exclusion column, and the results for the other sample components (e.g., DNA fragments, host cell proteins, amino acids, nucleotides, etc.) that were non-retained by protein A column are shown to the left, after the first application of the loading buffer and passage of these components through the size-exclusion column. Adapted with permission from Ref. .

Figure 4

(a) Sequence of an L-RNA aptamer that was used as a binding agent for L-tyrosine and related compounds and (b) chromatograms that were obtained when using this aptamer in an HPLC column for the chiral separation of D/L-tryptophan (top) and D/L-1-methyl tryptophan (bottom). Adapted with permission from Ref. .

Figure 5

Examples showing the use of HPAC to study biological interactions based on (a) competition studies in zonal elution experiments, (b) frontal analysis, or (c) ultrafast affinity extraction. The results in (a) are for the injection of L-tryptophan as a site-selective probe onto a 2 cm × 2.1 mm I.D. column containing immobilized HSA columns and in the presence of various concentrations of tolbutamide in the mobile phase (left-to-right: 20, 15, 10, 5, or 1 μM). The chromatograms in (b) were obtained for glimepiride that was applied to a 2 cm × 2.1 mm I.D. column containing HSA; the glimepiride concentrations were 50, 30, 20, 15, 10, or 5 μM (top-to-bottom). The data in (c) shows the effect of the injection flow rate on the measured free fraction for verapamil in a sample that contained a mixture of 10 μM verapamil and 20 μM AGP that was applied to a 0.5 cm × 2.1 mm I.D. AGP microcolumn. These figures are adapted with permission from Refs. , , and .

Figure 6

Examples of three stationary phases used in immobilized artificial membrane (IAM) columns based on phosphatidylcholine (PC). Reproduced with permission from Ref. .