Plasma Protein Binding assay
Aim: Screening of in vitro protein binding of test compound in plasma from human and/or preclinical species, using Rapid Equilibrium Dialysis (RED).
Background information: The extent of binding to plasma proteins influences the way in which a drug distributes into body tissues. High plasma protein binding limits the amount of free compound available to access sites of action and metabolism. This may impact on their therapeutic effects and elimination.
Equilibrium dialysis is the most widely accepted method for assessing plasma protein binding as non-specific binding effects are minimized compared to other methods such as ultrafiltration.
The RED device consists of disposable inserts and a base plate. Each insert is made of two side-by-side chambers separated by a vertical cylinder of a semi-permeable dialysis membrane with minimal nonspecific binding. The system is allowed to equilibrate at 37ºC. The amount of test compound present in each compartment is quantified by UPLC-MS/MS.
Compound requirement: 150 µl of a 10 mM DMSO solution
Turnaround time: 1 week / 1 compound / 3 species
- Test compound concentration: 5 µM
- Number of replicates: 3
- Equilibrium dialysis conditions: 37 ºC for 4 h
- Dilution with blank plasma (buffer sample) or buffer (plasma sample)
- Metabolic stability in plasma checked in parallel
- Analytical method: UPLC-MS/MS quantification of plasma and buffer samples after protein precipitation
- Percent of drug remaining in plasma
- Percentage of drug bound to plasma proteins
- Unbound fraction (fu) in plasma