The effect of sedimentation on the unbound fraction of therapeutic peptides in plasma determined using ultra-centrifugation method.

Abstract

Centrifugation is a critical step in determining the unbound fraction of drug (ƒu) in plasma, as it separates unbound drug molecules from protein-bound species during plasma protein binding (PPB) assays. When applied to therapeutic peptides, however, ultracentrifugation may induce vertical concentration heterogeneity within the centrifuge tube, potentially compromising the accuracy of ƒu measurements. In this study, we systematically investigated the primary factors contributing to concentration gradient formation for representative peptides spanning a wide molecular weight range. Our results identify molecular weight as the principal determinant of post-centrifugation sample heterogeneity. Compounds with molecular weights greater than approximately 2000 Da exhibited pronounced vertical concentration gradients, with differences greater than two-fold between the top and bottom regions of the supernatant. Notably, this phenomenon was observed in both human plasma and phosphate-buffered saline (PBS), demonstrating that gradient formation is driven by centrifugal force rather than plasma matrix interactions. These findings necessitate a critical methodological refinement for ultracentrifugation-based PPB assays. For compounds larger than ∼2000 Da, aliquots must be collected from a strictly defined mid-region of the centrifuge tube to ensure representative sampling. Failure to implement spatially standardized sampling introduces substantial quantitative bias and undermines assay reproducibility.