Potency Assays for Cell and Gene Therapy: A Complete Guide
Regulatory agencies such as the FDA, EMA, and ICH recognize potency as a critical quality attribute (CQA) for biologics. Developers must create consistent and reproducible quantitative assays to measure the therapeutic's specific biological activity or function.
At Forge Biologics, potency assay development is built on regulatory alignment, scientific rigor, and collaboration. Using a tiered approach, Forge tailors assays to each program’s stage and complexity, helping gene therapy developers meet global expectations while advancing confidently toward clinical and commercial milestones.
What Is a Potency Assay?
Potency assays for cell and gene therapy products are tests performed to measure the specific ability or capacity of the product to achieve the intended therapeutic effect. Potency assays are designed to quantify the biological function of the product in a way that is highly consistent and reproducible, allowing for release criteria to be set around appropriate parameters for therapeutic performance. These assays are primarily used to detect lot to lot variation between batches of the product and critically, allow for release or exclusion of tested materials.
Potency assays differ from all other types of tests performed on gene therapy vectors to measure safety, purity, infectivity, and identity. For example, vector titer measures the concentration of vector particles in a therapeutic and is a very important component in batch-to-batch variation between vector lots. However, potency is independent of titer. While vector titer reflects how many viral particles are present, potency measures what those particles actually do and the biological activity they produce. Two batches can have similar titers yet differ in potency due to variations in transduction efficiency or gene expression. Additionally, in vitro potency and TCID50 assays both rely on transduction of cells with gene therapy vectors, but the potency assays measure relevant biological activity, while TCID50 measures infectivity.
Most frequently, potency assays for gene therapy vectors are performed in vitro. In this type of assay, cells are transduced with vector and allowed to express the genes encoded within the vector. Scientists then measure cellular biological response to the vector to determine potency.
Why Potency Testing Is Critical for Gene Therapy Products
Potency testing plays a vital role in ensuring the safety, consistency, and efficacy of gene therapy products throughout their lifecycle. Because adeno-associated virus (AAV) and other gene therapy vectors are inherently complex biologics, even minor variations in production can impact therapeutic performance. Potency assays are therefore used to confirm that each manufactured batch retains the intended biological activity before release for clinical or commercial use. Beyond lot release, these assays are essential for monitoring product stability over time and establishing comparability when manufacturing processes, raw materials, or production sites change. Together, these data provide confidence that patients will receive a treatment that performs as expected, every time.
Regulatory agencies including the FDA, EMA, and ICH emphasize potency as a critical quality attribute (CQA) for biologics, codified in guidelines such as ICH Q6B, “Potency Assurance for Cellular and GT Products.” Regulators expect sponsors to develop quantitative, mechanism-of-action–based (MOA) assays that reflect the product’s intended clinical effect. Potency testing is not simply a box to check, it anchors the biological relevance of the therapy within a well-controlled, validated analytical framework. As gene therapies advance toward commercialization, robust potency assays become key to satisfying regulatory requirements, supporting license applications, and ensuring that every product released maintains its full therapeutic potential.
AAV Potency Assay Development: Key Considerations
Developing in vitro potency assays for AAV-based gene therapies is inherently complex because these tests measure how cells biologically respond to the vector. Since each gene therapy product has a unique design, target, and MOA, every potency assay must be customized to accurately reflect its intended therapeutic effect. The simplest assays measure quantification of vector-derived RNA or protein.
More advanced, MOA–based assays are designed to capture the therapy’s specific biological impact and often require more sophisticated experiments. These can include measuring downstream changes in gene or protein expression, enzymatic activity, cell morphology, or cell survival following transduction. Developing such assays requires careful selection of the cell model, optimization of transduction conditions, and thoughtful consideration of assay sensitivity and reproducibility.
Equally important is establishing robust reference standards and controls to ensure consistency across batches and over time. Developers must also define an appropriate analytical range and acceptance criteria to support assay qualification and eventual validation. Orthogonal potency assays can be used to support a potency assurance strategy through the potency lifecycle. The potency assurance strategy can target multiple potency CQAs. The goal is to design potency assays that not only reflect meaningful biological activity but can also be performed reliably and repeatedly under cGMP conditions, bridging scientific rigor with practical implementation.
The Potency Assay Development Lifecycle
The lifecycle of a potency assay typically includes three main phases: development, qualification, and validation. Among the suite of assays used to assess gene therapy safety, purity, infectivity, and identity, potency assay development generally requires the most time due to its bespoke nature. Each assay must be carefully optimized for a variety of variables to ensure it accurately reflects the therapeutic’s intended biological effect. Key considerations include:
- Cell Line – The cell line must be selected based on vector tropism and compatibility with the vector’s promoters and the therapy’s mechanism of action. Proper cell banking and testing of the cell bank are essential to ensure reproducibility and regulatory compliance.
- Experimental Readout – For nucleic acid-based assays, methods for extraction and amplification must be optimized to reliably detect vector-derived sequences. For protein-based assays, extraction and detection protocols must be carefully designed to capture the relevant biological activity. Other readouts may require detecting subtle or complex vector responses, often necessitating the implementation of new techniques or technologies.
- Analysis Method – The analytical approach depends on the type of experimental readout and the cellular response being measured. Acceptance criteria may evolve as additional data are collected, and assays progress through stages of clinical release, requiring robust statistical and modeling approaches to ensure consistency and accuracy.
AAV Potency Assays: Challenges
Potency assays for AAV vectors present multiple challenges, including variability between operators or lots, assay throughput, timeline pressures, cell density, promoter selection, transduction efficiency, gene expression variability, robustness, assay drift, sensitivity limitations, and statistical considerations. Addressing these challenges requires careful planning, iterative optimization, and thorough documentation throughout development, qualification, and validation phases.
Analysis Methods for Potency Assays
Several statistical models are commonly used to analyze potency assay data, each offering a different approach to determining relative potency. The parallel-logistic analysis method utilizes a three-, four- or five-parameter logistic regression model to generate a dose–response curve, allowing for precise calculation of relative potency between test and reference samples. The parallel-line analysis approach employs a linear regression model to evaluate relative potency based on parallel dose–response relationships across different concentrations. Finally, the slope-ratio analysis also uses a linear regression model. Each of these analytical methods can be applied depending on the nature of the assay, response curve, and degree of precision required to establish potency in gene therapy products.
Potency Assays in the CDMO Context
Integrating potency assay development with manufacturing is critical for ensuring efficiency, consistency, and regulatory compliance in gene therapy programs. In vitro potency assays typically progress through three phases: development, qualification (a subset of validation), and full validation. Process timelines allow challenges such as cell line selection, transduction optimization, and experimental readout to be addressed carefully.
When development moves at an accelerated pace, experienced operators must rapidly develop functional assays while building analytical models capable of adapting and improving as additional data become available. Iterative testing allows teams to model expected assay variability stemming from operator differences, testing locations, and lot-to-lot variation in reagents. By closely aligning potency assay development with manufacturing processes, CDMOs can ensure that assays are not only robust and reproducible but also practical for routine release testing, ultimately supporting smoother transitions from preclinical studies to clinical and commercial manufacturing.
What’s Next in Potency Assay Technology?
As the field of gene therapy continues to evolve, potency assay technology is advancing to meet increasing demands for precision, speed, and scalability. Emerging tools such as high-throughput assay designs are enabling researchers to analyze multiple samples and variables simultaneously, significantly reducing development timelines while increasing data robustness. In parallel, automation technologies are being integrated into potency testing workflows to minimize human error, enhance reproducibility, and streamline complex analytical processes. Together, these innovations are paving the way for more efficient, reliable, and scalable potency testing, critical for accelerating the development and commercialization of next-generation gene therapies.
How Forge Approaches Potency Assay Development
Forge takes a tiered approach to potency assay development, designed to align with each client’s stage of program maturity and regulatory needs. This method allows for early, scalable assay designs that can evolve in complexity as programs advance from research through clinical and commercial phases. Forge’s team provides comprehensive regulatory consultation and support, ensuring assay strategies meet global expectations from agencies such as the FDA and EMA. Central to Forge’s process is close collaboration with clients, combining scientific expertise with an understanding of each therapy’s unique MOA. As outlined in a recent Endpoints News feature, Three Tiers for Potency Assay Development Success, this integrated approach not only accelerates assay readiness but also strengthens regulatory confidence, ultimately helping gene therapy developers bring their products to patients faster.
Frequently Asked Questions
What is a potency assay in gene therapy?
A potency assay is a biological test designed to quantify the functional activity of a gene therapy vector, for example, its ability to produce the intended therapeutic effect. In the context of AAV therapies, potency assays help detect lot-to-lot variation, support release decisions, and ensure consistency in therapeutic performance.
How do you qualify a potency assay in gene therapy?
Qualification should be completed in time to support clinical investigations that are intended to provide substantial evidence of safety and efficacy for a marketing application. Drug Product release assays for a licensed product must be validated.
What’s the difference between potency and bioactivity?
Potency refers to the quantitative measure of the vector’s functional activity in achieving its intended biological effect. Bioactivity is a broader term encompassing any biological action or response elicited by the molecule; potency is a more specific, quantitative subset of bioactivity.
What are cell-based potency assays?
Cell-based (in vitro) potency assays involve transducing living cells with the gene therapy vector and measuring downstream cellular responses (such as RNA expression, protein production, enzyme activity, signaling changes, or morphological effects) that relate to the vector’s mechanism of action.
Can CDMOs help with potency assay development?
Yes. Many CDMOs (including Forge) offer potency assay development, qualification, and validation services, often with consultation and assay transfer support. Forge, in particular, uses a tiered potency approach to accommodate clients at different stages of development and helps technology transfer assays into their internal AD/QC teams.
I have my potency assay developed, can I tech transfer it to Forge?
In addition to developing potency assays, Forge also facilitates the technology transfer of existing potency assays from developers to our AD/QC teams. This approach is often used for early-phase programs that have already developed Tier 1 or 2 potency assays for in-house characterization of lots. These assays can be transferred to Forge for qualification and eventual use in clinical batch release testing. Late-stage programs may transfer fully developed and qualified potency assays for use in release testing. Check out our handy developer checklist for potency tech transfer readiness here.
