Optimization of Incubation Time for Accurate Protein Binding Assessment in Biological Matrices using Equilibrium Dialysis

introduction: Accurate estimation of the free fraction (fu) of a compound across biological matrices is critical for understanding pharmacokinetics, pharmacodynamics, and safety assessments in drug development. The use of equilibrium dialysis (ED) with relatively short incubation time (for example 2-6 hours) is not uncommon and may compromise the accuracy of fu determination. To evaluate the adequacy of commonly used incubation time in equilibrium dialysis (ED) for reliable protein binding (PB) estimation across diverse biological matrices and compound types. materials and methods: Time-course (2–24 h) of fu was conducted for seventeen marketed compounds with varying physicochemical properties using rapid equilibrium dialysis (RED) and HTDialysis devices across human plasma, 10 results: Tested compounds exhibited a wide fu range (<1>70%) and generally correlated with LogD as expected. Shorter incubation time was inadequate to reach equilibrium and could underestimate fu by up to 6-fold, particularly for highly bound compounds. discussion: Equilibrium was typically reached by 16–24 h in plasma, tissues, and cell lines, but faster in 10 conclusion: An incubation period of at least 16 hours is recommended for accurate fu estimation across biological matrices in early drug discovery. Routine assessment of fu in disease-relevant matrices is essential to support reliable in vitro–in vivo correlations (IVIVC) and scaling for pharmacokinetics, pharmacodynamics, and safety evaluations.