February 1, 2015 (Vol. 35, No. 3)

Jane Lamerdin, Ph.D. Director DiscoveRx
Helen Roberts, Ph.D. Director DiscoveRx
Abhishek Saharia, Ph.D. Director DiscoveRx

New Tools Allow Comparison of Efficacy, Safety, and Quality of Follow-On Biologics or Biosimilars

Biosimilars are defined as drugs that are “highly similar” to or “interchangeable” with an approved biologic; for example Celltrion’s version of Remicade, Remsima. 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. Some commercial cell-based assays for biosimilar development also offer greater physiological relevance to human biology than is achievable via traditional cell-based assays or animal models. 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, to incorporate commercial assays with said advantages or proceed with more physiologically relevant assays in complex human primary cell systems or animals.

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 has developed a wide array of such products that rely on native biology to directly interrogate therapeutic targets of currently marketed biologics. Figure 1 shows some of the over 30 well-validated PathHunter® assays for innovator drugs in diverse disease areas including diabetes (GLP1, Exendin, and Insulin), oncology (Bevacizumab), and inflammation (Anti-TNFα’s). The thaw-and-use, cryopreserved cell format of these assays offers greater operational flexibility as assays can be performed as and when needed without the requirement for cell bank creation. Ultimately such convenience improves the success of assay transfers to global testing and manufacturing sites.


Figure 1. Select examples of PathHunter bioassays. (A) Insulin bioassay demonstrates high reproducibility. (B) Bioassays for GLP1 and Exendin-4 use a cAMP readout. (C) Anti-TNFa bioassays enable benchmarking for biosimilars. (D) Simple PathHunter bioassay for Bevacizumab.

Case Study: Bevacizumab (Avastin®) Bioassay

To illustrate, validation 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 primary human umbilical vein endothelial cells (HUVECs) is an existing bioassay for bevacizumab. However, this assay requires >96 hours, utilizes primary cells that are difficult to maintain, and introduces performance variability due to changes in cell passage numbers, lots, and vendors.

The PathHunter VEGFR2 Dimerization Assay for bevacizumab 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 validated thaw-and-use cryopreserved cells, the PathHunter assay has many advantages over the standard HUVEC assay. Importantly, this assay is suitable for potency testing as determined in a multiday 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 antidrug antibody (NAb) detection.


Figure 2. 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 NHS.

In vitro Models of Human Safety and Efficacy

To demonstrate “similarity” to the innovator drug, there is also a clear need to assess biosimilar safety and efficacy by benchmarking the biosimilar against the innovator drug. For example, the development of biologics targeting proinflammatory cytokines such as TNFα has produced a significant advancement in rheumatoid arthritis therapy. The high global sales of these drugs (three of the top ten drugs in global sales are Enbrel®, Humira®, and Remicade®) and their limited patent life have made them high priorities for biosimilar development. In addition to the need for good potency and NAb assays for these drugs (Figure 1C), robust technology that provides a higher level of functional analysis to benchmark biosimilars in in vitro human models with predictive clinical outcomes prior to testing in the clinic offers not only a powerful tool to demonstrate “similarity” but can also provide discrimination.

BioMAP systems offer a fully optimized and automated human primary cell-based technology designed to model complex human disease and broad tissue biology in a practical, in vitro assay. In response to drug-versus-vehicle control, each system measures the relative levels of a set of translational biomarkers. These clinically relevant biomarkers, are combined to create a BioMAP profile with the potential to predict clinical outcomes for efficacy and safety, making it invaluable in comparing biosimilars to the innovator drug. As the biomarker profiles produced for different drugs in the BioMAP systems are robust, reproducible, and clinically relevant, biosimilar profiles can be benchmarked against the innovator drug or its variants (Figure 3B), or an entire class of biologics (Figure 3A). Altogether, the use of predictive in vitro systems like BioMAP can provide valuable information that can enable biosimilar benchmarking, help optimize production and formulation and provide useful predictive information on efficacy.


Figure 3. (A) Overlay of BioMAP profiles for Enbrel, Humira and Remicade in the BioMAP Diversity PLUS™ panel. All three drugs show highly similar biomarker profiles, yet some differences are apparent. (B) Bivalent Remicade and its monovalent Fab Remicade can be differentiated through an overlay of their BioMAP profiles.

Conclusion

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. The ability to benchmark biosimilars against the innovator drug in a predictive, in vitro primary human cell-based system, will provide a more systemic approach to predicting biosimilar efficacy and/or toxicity in human patients. 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.




























Jane Lamerdin, Ph.D., is director, R&D, Helen Roberts, Ph.D., is director of market development, BioSeek® division, and Abhishek Saharia, Ph.D. (asaharia@discoverx.com), is director of marketing at DiscoveRx.