Application of natural and modified biomacromolecules in miniaturised separative analytical techniques
In pharmaceutical R & D, drug stereochemistry, and consequently the rotation of enantiomers, is very important. Because they act as chiral selectors in vivo, biomacromolecules have been extensively used as chiral selectors for the liquid chromatographic (LC) resolution of enantiomers and more latterly have also been employed in the newer separative technique, capillary electrophoresis (CE). However, at the outset of this research programme, this had generally been restricted to common easily accessible biomacromolecules such as plasma-binding proteins. It was clear that it be would be useful therefore to adapt LC and CE in such a way as would allow the use of a much wider range of biomacromolecules. Accordingly the general aim of this study was to develop LC and CE protocols involving biomacromolecules that would give rise to minimum consumption of the biomacromolecule. To study biomacromolecules in free solution CE, a number of experimental variables had to be established for both optimum chiral discrimination and for investigating biomacromolecule-ligand interactions. The typical and widely used biomacromolecule for chiral discrimination, bovine serum albumin (BSA) was used to study the variables of pH from pH 5.4 to 8.4, concentration of BSA form 0 to 60 mM and concentration of organic modifiers in the range 0 – 20 % v/v for chiral selectivity. This involved an investigation into some unusual artefacts such as ghost peaks and stepped baselines, but ultimately the outcome was a successful free solution CE protocol suitable for the rapid evaluation of chiral discrimination of other biomacromolecules. The conditions were: run buffer (30 mM protein, 67 mM phosphate (pH 7.4) – methanol (97.5 : 2.5, v/v)), capillary CElect p150, 40 cm (35 cm to detector) x 50 mm i.d., temperature of ambient or 25 °C and an applied voltage of 10 kV. The ability of other biomacromolecules, such as human serum albumin (HSA), lactoferrin and protamine, to resolve enantiomers was studied using this protocol including looking at the effect of the addition of modifiers to the buffer such as metal ions like manganese and zinc, competing ligands, e.g. warfarin and ibuprofen, and b-cyclodextrin. As well as using CE, miniaturisation of LC was also studied in view of the success of biomacromolecule-affinity chiral LC. Two different, but similar, microbore LC protocols were employed, i.e. using the protein in free solution or as a pseudo stationary phase. For the former, a Lichrosorb DIOL stationary phase, based on hydroxyl groups immobilised on silica, was chosen in order to minimise the adsorption of protein to the stationary phase. Using this protocol it was demonstrated that free solution microbore LC could be easily be carried out, therefore used to evaluate chiral discrimination and that the use of the system to study in vivo interactions was feasible. The creation of a biomacromolecule pseudo stationary phase, as opposed to conventional chiral stationary phases where the protein is permanently bonded to the stationary phases, involves the biomacromolecule being adsorbed within the pores of the stationary phase. In this way the overall biomacromolecule structure should not be grossly distorted. Three stationary phases were evaluated, viz wide-pore Nucleosil silica, Nucleosil C8 and Lichrosorb DIOL, for optimum biomacromolecule loading and minimal biomacromolecule leakage when mobile phase was pumped through the column. The Nucleosil silica with adsorbed BSA proved the most successful, e.g. a of 3.6 and 4.0 for tryptophan and kynurenine respectively, and robust of the stationary phases with respect to demonstrating the chiral discrimination potential for this system. All the miniaturised systems evaluated were successful, to a greater or lesser degree, for the demonstration of chiral selectivity of biomacromolecules. While CE was better for minimisation of the consumption of the biomacromolecule, it was also important that the biomacromolecule LC systems could be operated in reduced dimensions since these systems have perhaps greater potential for exhibiting enantioselectivity and are more appropriate for the ever increasing need for the study of the interaction of ligands with the biomacromolecule in its ‘natural’ form. With the knowledge gained from this research programme it will now be possible to more easily carry out such studies with much smaller amounts of biomacromolecule, and, accordingly be able to work with biomacromolecules which hitherto it has not been possible to study because of limited availability. While some of the protocols have now been superseded by recent developments the system developed still has potential. The use of such small scale systems offers the potential to study chiral selectivity and drug-biomacromolecule binding of rare or expensive biomacromolecules.