Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay

Protein binding determination for highly-bound compounds using equilibrium dialysis remains a challenge in drug discovery. The reasons are mainly three-fold; 1. due to their slow diffusion rate, highly-bound compounds require a much longer incubation time to reach dialysis equilibrium than typically needed; 2. highly-bound compounds are often hydrophobic and prone to non-specific binding in dialysis; 3. free drug concentration in the post incubation dialysate is too low for reliable analytical quantification. Modified equilibrium dialysis approaches include using diluted plasma for dialysis, or pre-saturating the non-specific binding sites in the dialysis device with compounds of interest prior to dialysis. In this study, we developed a customized equilibrium dialysis assay with an extended incubation time of 24 h, followed by microflow (μF) LC-MS/MS for bioanalysis, for direct and definitive free fraction determination of highly protein-bound compounds. The extended incubation time ensured the dialysis to reach equilibrium and saturating the non-specific binding sites, while μFLC-MS/MS provided far better sensitivity than the conventional LC-MS/MS typically used for post incubation bioanalysis. For a group of commercially available, highly protein-bound compounds, the free fraction data generated by the developed assay correlated very well with the literature values generated with diluted plasma method or pre-saturation method. This novel assay approach has been successfully used to generate protein binding results for highly-bound compounds to support ongoing drug discovery research.