Chromatographic Studies of Protein-Based Chiral Separations

Abstract

The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha₁-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations.

Keywords: chiral high-performance liquid chromatography (HPLC); chiral recognition; chromatographic studies of drug-protein interactions; frontal analysis; protein-based chiral stationary phases; zonal elution.

Figure 1

Chromatograms obtained by zonal elution for the chiral separation of D- and L-tryptophan on high-performance liquid chromatography (HPLC) columns containing human serum albumin (HSA) immobilized to sulfhydryl-reactive silica that had been activated by using succinimidyl 4-(N-maleimidomethyl) cyclohexane-carboxylate (SMCC) or succinimidyl iodoacetate (SIC). Reproduced with permission from [35]. Copyright American Chemical Society, 2007.

Figure 2

Frontal analysis results obtained for R-warfarin on an immobilized human serum albumin (HSA). The breakthrough curves in (a) were obtained at 4 °C and using R-warfarin concentrations (from left-to-right) of 1.50, 1.30, 1.10, 0.76, 0.55, 0.33 and 0.22 μM. The plots in (b) were obtained when such data were acquired at various temperatures (4–45 °C) and analyzed according to a double-reciprocal form of Equation (3). Reproduced with permission from [65]. Copyright American Chemical Society, 1994.

Figure 3

Use of frontal analysis and band-broadening measurements to measure the effect of temperature on the (a) association equilibrium constants (K_a), (b) dissociation rate constants (k_d) and (c) association rate constants (k_a) for the interactions of D-tryptophan (■) and L-tryptophan (○) with a column containing immobilized human serum albumin (HSA). Reproduced with permission from [53]. Copyright Elsevier, 1997.

Figure 4

Examples of covalent immobilization methods for preparing protein-based CSPs on silica: (a) the Schiff base method and (b) the epoxy method.

Figure 5

Chiral separation of D- and L-tryptophan and analysis of the oxidation of D-tryptophan by the enzyme D-amino acid oxidase using a column containing immobilized HSA. Reproduced with permission from [87]. Copyright Elsevier, 2010.

Figure 6

Chiral separation of D- and L-3-cyclohexylalanine on a column containing immobilized anti-D-amino acid antibodies. Adapted with permission from [192]. Copyright John Wiley and Sons, 2006.