Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

• An entrapment method was developed for alpha1-acid glycoprotein (AGP).
• This method was used to make HPLC affinity columns for drug binding studies.
• The AGP columns were tested in both frontal analysis and zonal elution formats.
• Both entrapped AGP and human serum albumin were used to screen drug interactions.
• The measured binding constants agreed well with literature values.

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37 °C for carbamazepine with AGP was found to be 1.0 (±0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (±0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.