Entrapment of proteins in high-performance affinity columns for chromatographic studies of drug-protein interactions
Date of this Version
Poster Session, University of Nebraska-Lincoln Research Fair, April 4-5, 2017
The overall goal of this study is to develop a non-covalent immobilization process for quantitative studies of drug-protein binding, or related biointeractions, by high-performance affinity chromatography (HPAC). A series of polymeric monolithic supports based on glycidyl methacrylate-co-ethylene dimethacrylate (GMA-co-EDMA) with different porogen compositions were prepared. On-column entrapment was employed to immobilize human serum albumin (HSA) on monolith supports which had been converted into a hydrazide-activated and capped with oxidized glycogen. Warfarin and L-tryptophan were used as site-specific probes to evaluate preparation of these stationary phases through their binding with these solutes. Zonal elution studies with these materials also showed good agreement with the binding properties that have been reported for soluble HSA. This combined approach of using protein entrapment and HPAC gave a fast means for screening and studying drug-protein interactions.
On-column entrapment of proteins using monolithic support can be a suitable mean of investigating drug-protein interactions in HPAC.
The effectiveness of entrapment in immobilizing HSA within various monoliths, and the consequent change in drug binding, was seen to vary with the porogen composition that was used to make the monolith.
Lowering the percentage of 1-dodecanol vs. cyclohexanol in the polymerization mixture gave entrapped HSA supports with higher retention for drugs.
Since this immobilization method should result in little or no loss of protein activity, the data obtained in zonal elution studies such as used in this report should be useful in directly determining binding constants for drug-protein interactions in solution.
The future work will involve estimation of the moles of binding sites for each drug on HSA by also analyzing the support’s protein content. This analysis will make it possible to directly measure the association equilibrium constant for interactions of the drug with HSA. Frontal analysis of these columns will also be performed to evaluate the overall binding of drugs with proteins in these supports.