Plasma Protein Binding Assay

Pharmacokinetic and pharmacodynamic properties of test compounds are affected by the unbound (free) plasma concentration, as compound bound to plasma proteins is unable to reach an effective concentration at the site of action. It is highly important to understand the degree of plasma protein binding (PPB) as this not only impacts the efficacy of your drug candidate, but also influences potential toxicity. Domainex assesses test compound plasma protein binding by equilibrium dialysis using Rapid Equilibrium Dialysis (RED) devices. 

Domainex’s Standard Experimental Procedure:

Plasma protein binding is measured by Rapid Equilibrium Dialysis method, using RED device inserts. Test compounds and assay standards (a highly bound and a medium bound compound) are incubated in plasma and phosphate buffered saline (PBS) at 37 °C for 4 hours in different chambers in the RED device separated by a semi-permeable membrane. After this time, samples from each chamber are matrix balanced and proteins precipitated using organic solvent. Samples are diluted further for Ultra-High Performance Liquid Chromatography (UHPLC)-Mass Spectrometry (MS) analysis. Example data is shown in Figure 1.

Figure 1: Example fraction unbound results for four test compounds and a control (warfarin) in different species. 

*Other options available upon request

Data Analysis:

Extracts are analysed using Waters Acquity UHPLC TQ-S, TQS-Micro or TQ-XS instruments. Triple quadrupole mass spectrometers, operated in multiple reaction monitoring (MRM) mode, provide accurate quantification with excellent sensitivity, selectivity and reproducibility.



The results are reported in Excel file format as compound bound (%), compound free (%) and recovery (%) including standard error of mean (SEM). Any relevant comments about compound stability/solubility/binding are also included in the report.

Turnaround time from receipt of the compounds to release of data is typically less than 2 weeks.