One of the major bottlenecks in the development of biosimilars is the need for good bioassays to create potency,
stability and neutralizing antibody (NAb) assays. Ideal bioassays need to reflect the clinical mechanism of action
(MOA) of the originator drug and should be simple, precise, reproducible and robust. In addition, serum/matrix
tolerance and sensitivity are extremely important factors in the development of quantitative NAb assays. Avastin
(bevacizumab), a 149 kDa humanized IgG1 monoclonal antibody that selectively binds and inhibits vascular endothelial growth factor A (VEGF-A), is a particularly challenging product for biosimilar assay development, as relevant functional readouts can be time-consuming and require handling of finicky HUVEC cells.
Here, we discuss the development and application of PathHunter® cell-based assays that rely on the native biology
of the VEGF receptor (VEGFR), thereby reflecting the clinical MOA of the originator drug. Specifically, the Avastin
assay quantifies VEGFR2 homodimerization upon binding of VEGF-A, which is blocked by addition of Avastin or
appropriate biosimilar molecules.
This assay is highly specific, robust and utilizes a homogenous mix-and-read protocol, facilitating rapid and reproducible quantitation of drug potency. The technology also enables accurate and sensitive detection of neutralizing antibodies even in high concentrations of human serum through a simple chemiluminescent output. The assays are developed in a convenient ready-to-use format that minimizes assay variability that may result from cell culture. The cell preparation, bioassay protocol and reagents have been optimized to provide superior bioassay performance with high reproducibility (<7% RSD).
“Avastin®” is a registered trademark of Genentech, Inc. (USA).