By Jane Lamerdin, Ph.D., Abhishek Saharia, Ph. D. DiscoveRx Corporation, Fremont, CA 94538, USA
Biosimilars are defined as drugs that are “highly similar” to or “interchangeable” with an approved biologic. The lack of a clearly defined path to demonstrate such biosimilarity leaves the burden of proof on developers. Showing efficacy, quality, biopotency and clinical safety often requires a panel of assays, including analytical, biochemical, cell-based and even in vivo assays. Cell-based assays offer significant advantages over in vivo studies, offering lower resource requirements and cost, with improved speed and performance characteristics. We discuss here some examples and how they help reduce internal costs and timelines, as well as offer excellent reproducibility across global sites.
Biosimilar developers face challenging decisions in determining the best pathway to proving the biosimilarity of their product. These include whether to develop bioassays in-house or to incorporate commercial assays with said advantages. An ideal potency bioassay should mimic the mechanism of action (MoA) of the innovator drug while producing highly precise, accurate and reproducible data in a quality (GLP or GMP) environment. Traditionally, bioassay developers have adapted cell proliferation and live animal assays that reflect events occurring well downstream of the drug target, with poor target specificity. These phenotypic assays suffer from lengthy, complex protocols, high variability and/ excessive cost. Furthermore, assays reliant on immortalized cell lines must incorporate additional resources for cell banking and continuous performance monitoring due to potential cellular drift. Commercial cell-based potency assays greatly reduce the time and cost of assay development and validation as they are ready-to-use, well-qualified and often easy to adopt. DiscoveRx (www.discoverx.com/biosimilars) has developed a wide array of such products that rely on native biology to directly measure functional potency of currently marketed biologic drugs, with the goal to aid development of biosimilars.
Case Study: Bevacizumab (Avastin®) Bioassay
To illustrate, qualification of a potency assay for the angiogenesis inhibitor bevacizumab is shown. VEGF-A activates VEGFR2 through receptor dimerization, promoting proliferation of endothelial cells. Bevacizumab binds to and inhibits VEGF-A; thus proliferation in human umbilical vein endothelial cells (HUVECs) is an existing bioassay for bevacizumab. However, this assay requires >96 hours to run, utilizes cells that are difficult to culture and introduce performance variability due to changes in passage number, culture conditions and operator. The PathHunter® Bevacizumab bioassay quantifies the inhibition of VEGF-A-induced dimerization of the VEGFR2 receptor. As shown in Figure 1D, the PathHunter assay generates data that is consistent with the HUVEC proliferation assay (VEGF-A: ED50 of 1-6 ng/ml; bevacizumab: ED50 of 50 ng/ml). Additionally, with its shorter assay time (<24 hours), simple ‘add and read’ protocol and cryopreserved thaw-and-use cells, the PathHunter assay has many advantages over the standard HUVEC assay.
Importantly, this assay is suitable for potency testing as determined in a multi-day qualification exercise (Figure 2A, B), where the assay displayed very good accuracy (95.9%), excellent precision (4.1%), a relative standard deviation (RSD) <5%, and good linearity (r2 =0.985). Additionally, this assay demonstrates excellent matrix tolerance of up to 90% normal human serum (NHS) with little change in EC50 values or signal to background ratios (Figure 2C), enabling the use of this assay for neutralizing anti-drug antibody (NAb) detection when testing for clinical immunogenicity of the biosimilar. As innovator drugs come off patent and global biosimilar development activity increases, the need for specific tools to accelerate and reduce costs of biosimilar development is evident. Commercially available, ready-to-use cell-based assays will enable biosimilar developers to rapidly advance to the critical assay validation stage for potency and NAb assays. Companies that are the quickest to demonstrate “similarity” of their molecule using available commercial tools can hope to be the first to market with their biosimilars.
Here we tested the PathHunter VEGFR2 homodimer assay with VEGF-A, demonstrating a robust and reproducible response A. The VEGFR dimerization assay was tested with four test samples, from 50% to 150%, compared to a reference standard (100%). B. The measured relative potency was plotted against the expected relative potency. C. Matrix effect on VEGFR2 bioassay was evaluated by addition of small volumes of normal human serum (NHS).