Publication Date
2012
Document Type
Dissertation
Committee Members
Gerald Alter (Committee Member), Roger Gilpin (Advisor), Daniel Ketcha (Committee Member), Richard Sherwood (Committee Member), Kenneth Turnbull (Committee Member)
Degree Name
Doctor of Philosophy (PhD)
Abstract
The pharmacokinetic difference between drug enantiomers is the impetus for developing analytical techniques to assess enantiomeric purity. Capillary electrophoresis (CE) is an analytical technique that is used for characterizing drug-protein binding. The pitfall to using CE for drug-protein binding studies is protein chiral selectors tend to adsorb onto capillary walls and cause changes in electroosmotic flow that lead to decreased enantioselection and migration time irreproducibility between consecutive injections. The experimental parameters for minimizing the adverse effects of protein adsorption are not clear from the literature.
Rinsing protocols to improve enantioselection and migration time repeatability were developed using the tryptophan-bovine serum albumin system as a model. The enantioselection of bovine serum albumin (BSA) could be improved by: 1) increasing separation voltage; 2) using sample buffer ionic strength at least 3 orders of magnitude less than the separation buffer; 3) limiting the equilibration time with separation buffer; and 4) allowing for protein diffusion. Rinsing the capillary with sodium hydroxide, followed by water improved migration time repeatability RSD from 24.7% to 1.8% (n = 4). Drug-protein binding is contingent upon the three dimensional structure of the binding site, and the presence of other competing drug molecules. Drug-drug displacement is difficult to predict and the effects of protein glycation on binding of drugs is not well defined. To highlight the use of CE for addressing questions of biochemical interest, CE was applied to characterize drug-drug displacement and the effects of protein glycation on the enantioselection of drugs by BSA. The tryptophan-bovine serum albumin, 5-fluoro-tryptophan-bovine serum albumin, and 5-hydroxy-tryptophan-bovine serum albumin systems were used as models.
A CE method for studying competitive binding was established using ibuprofen as the displacer molecule. Accurate calculation of selectivity was found to depend on the precomplexation of ibuprofen and BSA. A CE method for studying the effects of protein glycation was developed using BSA containing different degrees of glycation as chiral selectors. The enantioselection of tryptophan analogs by BSA was altered by glycation as reported in other analytical methods. These studies can serve as guidelines for optimizing serum albumin enantioselection and extending its use in other biopharmaceutical applications.
Page Count
191
Department or Program
Biomedical Sciences
Year Degree Awarded
2012
Copyright
Copyright 2012, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
