Protein Binding

The binding of test compounds to plasma proteins is an important parameter for metabolism and pharmacokinetic studies. If the drug is highly bound to plasma proteins, the amount of drug available is reduced, and subsequently the efficacy may be significantly reduced. Determination of the level of protein binding is critical and may be helpful in correlating with in vivo efficacy. We routinely use a rapid centrifugation technique to determine plasma protein binding, but also have available a rapid equilibrium dialysis device that correlates with the more traditional equilibrium dialysis approach.

Centrifugation
A solution of test compound in plasma is prepared and incubated for a pre-determined time period. After incubation, the test samples are transferred to an ultra filtration device equipped with a size exclusion membrane and centrifuged. The membrane allows small molecular weight compounds through: however, it retains large proteins. Any test compound that is bound to a large protein will not pass through the membrane. The filtrates are analyzed for test compound concentration by LC/MS/MS and compared to a PBS control treated in the same manner.

Rapid Equilibrium Dialysis Device
Plasma spiked with test compound and added to the center chamber of a commercial plate based RED (rapid equilibrium dialysis) device. Blank, isotonic sodium phosphate buffer is added to the peripheral chamber of the RED device and the plate is incubated at 37°C . Aliquots of the buffer and the plasma are taken at pre-determined time points and the concentration of free and bound test compound is determined by LC/MS/MS analysis.