Discussion

Cell-based bioassays often pose a hurdle during a rapidly moving biologics development program. High standards
for assay accuracy, precision, reproducibility and robustness are additionally put to the test by the use of continuous
culture cells that can add to variability and increase the cost and complexity of each assay. This is particularly
challenging for anti-VEGF drugs, as the prevalent assay is the proliferation of human umbilical vein endothelial cells
(HUVECs), which requires 72-96 hours to run, utilizes cells that are diffi cult to culture and introduces performance
variability due to changes in passage number, culture conditions and operator. Here, we describe the PathHunter®
bioassay that has been developed as a fi t-for-purpose QC Lot Release assay for anti-VEGF drugs. The assay quantifi
es inhibition of VEGF-A-induced VEGFR2 receptor activation, by measuring receptor dimerization as an early event
in the receptor activation cascade. With its shorter assay time (<24 hours), simple ‘add and read’ protocol and use
of cryopreserved ready-to-assay cells, the PathHunter assay has many advantages over the standard HUVEC assay.
Data will be presented comparing the performance and reproducibility of the PathHunter Bevacizumab bioassay to
the standard HUVEC proliferation assay.