Precision in affinity capillary electrophoresis for drug-protein binding studies

In order to achieve excellent precision in the estimation of binding constants by affinity capillary electrophoresis (ACE), electroosmotic flow (EOF) stability is the key parameter, especially when using proteins in binding assays. Appropriate rinsing protocols are mandatory. In our study, the capillary was rinsed after each run with 0.1 mol/L sodium hydroxide for 2.0 min, with water for 2.0 min followed by running electrolyte (phosphate buffer at pH 7.4) for 3.0 min (pressure=3000 mbar each). Tryptophan-human serum albumin, warfarin-bovine serum albumin and quercetin-beta-lactoglobulin were used as ACE models. Further improvements in precision have been obtained by avoiding a complete standstill of liquid within the capillary and flushing the capillary with buffer for 25 min after each 30 consecutive runs. The precision of measurements is further improved by the use of mobility ratios to report mobility changes (RSD% less than 0.5% in a long-term measurement, n=300-600). Apart from the importance of a stable EOF, other ACE key parameters include protein concentration, drug plug length, applied voltage, and the choice of the regression method. In the present work, useful protocols and templates are provided in order to allow users a quick and efficient start with ACE methods. The comprehensive experimental part can serve as a checklist, which parameters need to be addressed for successfully applying ACE. Here, the suggested experimental design allows for the determination of binding constants within a couple of hours using standard instrumentation. This time could still be decreased by orders of magnitude using capillary arrays or miniaturized systems.