Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs

J Pharm Sci. 2011 Jun;100(6):2498-507. doi: 10.1002/jps.22452. Epub 2011 Jan 6.


Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.

MeSH terms

  • Blood Proteins / metabolism*
  • Chromatography, Liquid
  • Dialysis / instrumentation
  • Dialysis / methods
  • Drug Discovery* / instrumentation
  • Drug Discovery* / methods
  • Drug Interactions
  • Drug Stability
  • Humans
  • In Vitro Techniques
  • Limit of Detection
  • Pharmaceutical Preparations / blood*
  • Pharmaceutical Preparations / chemistry
  • Reproducibility of Results
  • Solubility
  • Tandem Mass Spectrometry


  • Blood Proteins
  • Pharmaceutical Preparations