Fig. 4.

(A) Schematic illustration of the workflow for the PPB assay. (a) Samples are provided from the same source plates as those used for the logD7.4 and Sol assays. (b) Compounds are pooled in a pool plate on a Hamilton STARplus. (c) Compound pools are transferred to plasma-containing deep well plates (referred to as standard plates), mixed by shaking and centrifuged. Part of content is further diluted in an intermediate plate, while remaining content is serially diluted to create standard curves (top). Content in the intermediate plate is mixed by shaking and centrifuged before transfer of samples and buffer to the inward and outward chambers of Red Devices, respectively (bottom). (d) Time zero samples and standards are transferred from the intermediate plate to crash plates containing AcN with IS and matrix matched samples. The crash plate is kept in the freezer overnight. Incubated buffer and plasma samples from the Red Devices are transferred to the crash plate alongside plasma stability samples (T18) from the intermediate plate. The crash plate is thoroughly shaken and centrifuged cold. (e) Samples are diluted in acidified H₂O and subsequently in acidified 37% AcN to and detected using UPLC-MS/MS. (B) Correlation between data in the automated PPB assay versus literature data for 47 drugs. The slope between average values from a minimum of three independent measurements with previously reported data is 0.964 (r² = 0.834). Dotted lines represent the 95% CI 0.835–1.09. (C) Correlation between two independent experiments submitted to the database. When more than two independent replicates were available, we included the first two instances. The slope based on testing of 624 compounds comes out at 0.982 (r² = 0.960) with a 95% CI of 0.966–0.997. (D) Correlation between harmonized assays at AstraZeneca Gothenburg (y-axis) and at Pharmaron (x-axis). The comparison is based on 801 compounds with a slope of 0.977 (r² = 0.938) and a 95% CI of 0.960–0.994. (E) Manhattan plot following 6 years of operation from May 2013 to June 2019—warfarin (brown filled circle), metoprolol (green filled triangle), and propranolol (black filled diamond). Color images are available online.