A potency or potency-indicating assay is a regulatory requirement for the release of every lot of a biotherapeutic. Potency is a critical quality attribute that is also monitored as a stability indicator of biotherapeutics. In the absence of full product understanding, the biologics world is still bound by the concept of “the process defines the product.” For complex biologics, manufacturing processes may be optimized to yield what is believed to be an active ingredient, but a company may end up, sometimes unknowingly, with reduced activity due to removal of a key component. This is where meaningful assays play such an important role. Changes in potency can occur through a variety of physicochemical or structural changes. A meaningful and reliable assay is probably the most important tool in drug development. 

Protecting bioassay-based estimates of relative potency against bias (due to allowed non-similarity) and against unacceptably high similarity failure rates, while allowing for changes in assay capability (precision) appears to be impractical. While power calculations for both detecting non-similarity (via difference tests) and for passing similar samples (via equivalence tests) are particularly helpful, these calculations are not simple. This presentation will illustrate some tools for these calculations and how they support modifying similarity criteria based on data from bioassay.

Potency is a critical attribute in assessing the quality of clinical-grade products during process development, GMP release, and stability monitoring. Cell-based potency assays are essential to demonstrate the mechanism-of-action of drug products in clinical trials for regulatory approval. Here, we discuss the strategies and challenges of the development and qualification of a functional potency assay to support the release of gene therapy products and biologics. Two case studies will be used to demonstrate how to simplify the method by making it more robust, less time-consuming, easy to maintain, and requiring fewer critical reagents.

An immunotherapy that binds to ILT3 can block the ligand-ILT3 interaction, reversing an inhibitory signal cascade. A novel chimeric receptor reporter cell-line was designed and engineered that inverted ILT3 from an inhibitory to an activation motif, ITIM to ITAM to measure drug-induced regulation of gene transcription. This cell-based potency assay enabled anti-ILT3 drug disruption of ligand interaction and was sensitive to anti-ILT3 drug stability stresses.

The manufacturing and release of cellular therapy products (CTPs) requires high-quality, robust, and validated analytical methods. Here I will describe recent developments in analytical method standardization, including the FDA standards recognition program and NIST public-private partnerships to support the development of critical analytical methods. A key aspect of analytical development for this new class of products is the need for a fit-for-purpose approach. Efforts to identify and establish fit-for-purpose process analytical technologies will also be described.

Sponsors must develop a potency assurance strategy early in drug product development. The recently issued FDA draft guidance on potency assurance of CGT products will be discussed, along with a risk-based approach to developing a product potency assurance and bioassay development strategy.

Advanced therapies such as CAR T cells are complex living drugs with multiple mechanisms-of-action. Due to this complexity, the strategy for developing and qualifying a single potency assay that correlates with efficacy has been challenging. In this talk, we will address the pros and cons of various assays commonly used, as well as the advantage of developing these assays in early clinical development.

The United States Pharmacopeia (USP) General Chapter <1033> provides guidance on the validation of biological assays. We highlight key statistical considerations in the interpretation of this guidance, including setting acceptance criteria based on the probability of out-of-specification results and the use of total analytical error to quantify accuracy and precision. These reflect an evolving approach towards bioassay validation, underscoring current best practices and continual improvement.

Subcutaneous (SC) delivery can exacerbate immunogenicity of biologics where injected protein is exposed to skin-derived dendritic cells (DC) migrating into the injection site and toward lymph nodes. In a newly developed, mechanism-based dendritic cell migration assay, chemokine receptor expression on DCs and their in vitro migration correlated strongly with clinical immunogenicity incidence. This assay can inform decision-making of SC immunogenicity risk, with applicability to screen formulation and quality attributes.

When developing a biotherapeutic, the risk of immunogenicity is a crucial factor to consider during the lead selection phase. To better understand, manage, and minimize the potential impact of immunogenicity on the clinical tolerance and activity of drug discovery, we've established a set of strategies by using a suite of in-vitro tools for immunogenicity risk assessment and mitigation during therapeutic lead identification and optimization. I will share a case study demonstrating the practical application of our multiple tools in assessing immunogenicity risk. Through this case study, you will gain insights into how these tools can effectively be used together to drive decision-making for therapeutic lead selection.

All approved ADCs have cell-killing NAb assays and industrial consensus is consistent with this. Multiple MOAs exist in vivo for ADCs; however, in vitro NAb assays can only monitor target-involved internalization and killing. In addition, artifacts may exist for in vitro cell-killing NAb assays, for example, FcgR-involved non-specific killing. We thus propose that like mAb therapeutics, cell-binding or competitive ligand-binding NAb assays could be considered for ADC.