Determination of Plasma Protein Binding by Ultrafiltration

Purpose

As a criterion in an ADMET discovery lead evaluation program, the degree of binding of a compound to plasma proteins can provide important indications about the compound's efficacy and its potential for drug-drug interactions. A rapid technique to determine plasma protein binding is ultrafiltration centrifugation.

Assay Protocol

Plasma pools from each required species are fortified with test article at two appropriate concentrations, in duplicate, and pre-incubated at 37°C for approximately 15 minutes. The spiked plasma is then transferred into Centrifree^® ultrafiltration devices and spun at 1000 g for 20 minutes. Portions of the resulting ultrafiltrates are analyzed for test article concentration by an appropriate method of analysis (LSC, HPLC or LC/MS/MS) and the degree of binding to the plasma proteins calculated. A portion of phosphate buffered saline (0.1M, pH 7.4) is treated in the same manner and the recovery of the test article from the apparatus estimated, thus indicating the potential for non-specific binding during the investigation.

Confirmation Data

The degree of binding of a selection of compounds with various binding properties to mouse, rat, dog, monkey and human plasma proteins was determined by ultrafiltration with LSC and/or HPLC analysis. The results obtained agree well with those quoted in the literature and confirm the validity of the method adopted at Ricerca. In addition, the results yielded by LSC and HPLC were similar confirming the acceptability of the use of nonlabeled material combined with HPLC analysis.

The Degree of Binding of a Selection of Compounds to
Plasma Proteins of Various Species as Determined by LSC or HPLC