Plasma Protein Binding

Drugs may bind to a wide variety of plasma proteins, including albumin, and the degree of binding can impact on the pharmacokinetics and pharmacodynamics parameters of a drug, given that only the free drug in plasma is available to elicit a pharmacological effect and only the free drug is available to be cleared. Hence it is highly important to measure plasma protein binding during the drug discovery phase.

The leading approach for assessing plasma protein binding is rapid equilibrium dialysis, as the impact of non-specific binding is minimised when compared to other methods such as ultrafiltration and HT-Dialysis, which are relatively slow processes to reach equilibrium.

Sygnature’s Plasma Protein Binding assay utilises the Rapid Equilibrium Dialysis (RED) device to measure the percentage binding of a test compound to plasma proteins in a variety of species (e.g. human, rat, dog, mouse, monkey). This allows the percentage unbound to be calculated. Test compounds and positive controls (species dependent) are incubated in 100% plasma and dialysed against buffer in a RED device for 4 hours at 37°C in a 5% CO₂ incubator, with continuous shaking at 200 rpm. Samples are matrix matched and analysed by LC-MS/MS against a standard curve prepared with 100% plasma.