Potency Under the Microscope: What Regulators Expect from RNA and Gene Therapies Today

April 24th, 2025

The growing attention to viral and RNA-based biologics, from gene therapies to mRNA vaccines, has impacted the therapeutic landscape. These modalities share an underlying mechanism that is action based on delivering genetic material to host cells, enabling in situ expression of therapeutic proteins or antigens. While viral vectors (e.g., AAV, AdV, lentivirus) use modified viruses to carry DNA into cells, RNA-based products (e.g., mRNA vaccines) rely on direct delivery of messenger RNA, often encapsulated in lipid nanoparticles.

Potency plays a central role in ensuring these therapies are safe, effective, and consistently manufactured. A well-characterized potency assay is not just a regulatory checkbox; it’s a cornerstone of product development, release, and lifecycle management.

Regulatory expectations around potency have evolved rapidly due to key scientific advances, lessons from accelerated development pathways (notably during the COVID-19 pandemic), and increased commercial interest in these modalities.

In today’s blog, we explore how these expectations have changed and what it means for developers.

 

Historical Context

Historically, potency testing for biologics, like vaccines and monoclonal antibodies, focused on relatively well-understood and standardized assays, often in vivo models or simple binding/neutralization tests.

When viral vectors and RNA-based therapies entered the scene, initial regulatory strategies attempted to retrofit traditional potency views. However, this quickly proved insufficient. These novel modalities presented unique challenges, such as complex intracellular mechanisms of action, high sensitivity to degradation, and multi-step pathways from delivery to protein expression.

In the case of gene therapies, early trials relied on vector titers as proxies for potency, while mRNA vaccine developers often used in vitro expression or immunogenicity in animal models. These were first steps on the path, shaping the current approaches to potency in such complex biologics. These data were valuable, but not always reflective of the actual clinical performance.

 

Current Regulatory Landscape

Regulators now recognize the unique requirements of these products and have adapted their expectations accordingly.

FDA (CBER/CDER): The FDA emphasizes mechanism-of-action (MoA)-reflective potency assays, particularly for gene therapies. Draft guidances encourage early development of cell-based functional assays and discourage sole reliance on surrogate measures.

EMA: Similarly, EMA expects potency assays to be quantitative, robust, and directly linked to the product’s biological activity. There is increasing attention on the need to define potency in early development and maintain it throughout lifecycle changes.

ICH Q6B: While this guideline provides a broad framework, interpretation for novel products often requires additional interaction with regulators.

Both product-specific guidance (e.g., for mRNA COVID-19 vaccines) and emerging platform-based thinking are shaping the regulatory narrative.

 

Challenges in Defining and Measuring Potency

Potency measurement for viral and RNA-based biologics remains a scientific and regulatory challenge:

• Mechanistic complexity: These products often involve multiple steps from delivery to expression to action, complicating assay design.
• In vivo models not available or adequate (e.g. transgenic animal models are accepted for research and Proof of Concept stages, but not usable for release & stability testing due to lack of robustness,focus of replacement of routine in vivo testing, and are expensive and time-consuming)
• Assay variability: Cell-based assays can be prone to variability, especially with primary cells or transient transfections.
• Comparability: Establishing consistency between lots or across manufacturing changes is difficult without a stable, well-characterized potency method.

Key Assay Types & Trends

Given these challenges, a range of assay strategies has emerged:

• Cell-based functional assays: Reflect biological activity (e.g., reporter gene assays, transduction efficiency in gene therapy).
• Analytical surrogates: Tools like RT-qPCR, ELISA, or HPLC may be used to measure encapsulated RNA or expressed protein levels.
• Orthogonal/matrix approaches: Combining multiple assays offers a fuller picture of potency and helps mitigate limitations of individual tests.

The trend is toward mechanistically relevant, reproducible assays that correlate with clinical performance.

Potency in the context of CMC and Lifecycle Management

Potency is not a standalone metric; it is tightly linked to the overall Chemistry, Manufacturing, and Controls (CMC) strategy:

• CQAs and control strategy: Potency must be tied to a critical quality attribute (CQA) and integrated into the control strategy, including the attention to particular assay’s ability to satisfy the qualification criteria for release purpose and stability indicating and comparability demonstrating needs.
• Process changes: Bridging studies must demonstrate that potency remains unchanged after changes in cell lines, upstream/downstream processing, or formulation.
• Developmental phases: Regulatory expectations intensify from Phase 1 to commercial approval. Potency methods must evolve accordingly and remain stable through tech transfer and scale-up.

Looking Ahead: Future Directions

As the field matures, regulatory and scientific communities are exploring new paradigms:

• Platform analytics: Especially for RNA products, standardized methods may reduce the burden of product-specific development.
• In silico and systems biology models: These may support or complement traditional potency assays.

• Dual testing approach: parallel development and application of Transgene functionality, which is relying on detection of quantitative signals of biological activity, as close as possible reflecting Mechanism of Action and orthogonal Mechanism of action characterization assay, which is covering end to end steps of processing of genetic information, before transgene is involved in mechanism of action. The first assay needs to be fit to release, stability and comparability testing use and second assay can be characterization of end to end genetic functionality of encoded information and support Proof on concept studies and monitoring needs.

• QbD integration: Potency will increasingly be viewed through a quality-by-design lens, with focus on understanding variability and establishing design spaces. In the upcoming FDA CGT Potency guidance (draft version – review/consulting phase is finished) – the concept of Potency assurance strategy is emphasized. It is a multi-angle approach that reduces risks to the potency of the product through design of manufacturing process, in-process control strategy, incoming materials control, and release assays to release the final lot. Complete strategy must include multiple assays, for different purposes: release, characterization, stability proof of concept, etc.

Key Takeaways

• Potency remains a central and evolving concern for viral and RNA-based biologics.
• Regulators expect mechanism-based, quantitative, and robust potency strategies, which includes several potency methods, fit for intended use.
• Developers should engage early with agencies to align on  different assay expectations and develop several assays in parallel.
• Orthogonal approaches and lifecycle-minded assay development are critical to success.

 

Have you encountered challenges in developing potency assays for viral or RNA-based products? What strategies have worked for your team?

Share your experiences or reach out to connect. We’re always keen to collaborate and learn from fellow innovators in the field.

 

Authors: Dmitrii Sorokin, Head of CMC, Exothera and Hanna Lesch, Chief Technology Officer, Exothera

 

About Exothera

Exothera is a specialized CDMO partner delivering preclinical and clinical process development, manufacturing optimization and CMC support for virus and RNA based therapeutics and vaccines.

With headquarters in Belgium, at the heart of Europe, we assist clients worldwide from start to scale, up to commercial manufacturing, including analytical testing and regulatory consultation. Thanks to our optimal technologies and experienced team, we help bring innovative therapies from bench to patient with quality, at speed and cost-effectively.