Potency assays are essential for demonstrating that biotherapeutics perform their intended biological function throughout development and commercial manufacturing. Selecting the right assay format requires balancing biological relevance, analytical robustness, and regulatory expectations. This blog explores the different types of potency assays, key development considerations, and best practices for designing fit-for-purpose methods that support product quality and regulatory compliance.
For biotherapeutics, potency assays play a central role in the analytical strategy. They are not simply another release test. Their purpose is to show whether the molecule can perform the biological function it was designed to deliver. A well-chosen potency assay therefore has to reflect the mechanism of action (MoA), the underlying product biology, and the expected clinical relevance, while still being practical enough for routine quality control (QC) use.
Potency is considered a critical quality attribute (CQA) because any shift in biological activity can influence clinical performance, safety, and batch-to-batch consistency. A potency assay has to reflect the biology of the product, but it also needs to perform reliably in routine QC use. Depending on the MoA, the most appropriate format may be a cell-based assay, a binding assay, an enzymatic assay, or a surrogate assay. Each option has value, but each also has limitations. Cell-based assays may be closer to the product’s biological activity, while simpler binding or surrogate formats may be easier to control. In practice, assay selection comes down to finding the best balance between biological relevance, precision, reproducibility, and practical performance.
This is why potency assay work should begin early in the product lifecycle. Early planning gives teams time to compare different assay formats, identify the most relevant and robust option, and generate data on stability, consistency, and potential links with clinical efficacy. When direct measurement of biological activity is difficult or not feasible, this early work is also important for showing whether a surrogate assay can reliably reflect the product’s biological function.
Why potency is central to biotherapeutic development
Potency is watched closely from early development through commercial quality control. For batch release, it confirms that each lot exhibits the intended biological activity, supporting decisions regarding clinical or market readiness. In stability studies, potency assays show whether product activity is maintained over time, as even a minor drift can indicate degradation, aggregation, or a process-related shift.
During comparability assessments, potency assays address a direct question: does the product maintain its biological performance after a process change, site transfer, scale-up, or formulation update? In this context, potency assays go beyond the routine quality control (QC) check; they provide evidence that product performance remains consistent across lots and across the product lifecycle. Regulators look closely at the assay format, controls, reference standard, and acceptance criteria. Potency assays occupy a unique position: while it is an analytical method for a biotherapeutic, it also carries a line of sight to clinical relevance. More than a test, a potency assay serves as a bridge between product quality and patient impact.
Types of potency assays
Potency assays can take several forms, as no single format fits every biotherapeutic. Selecting the appropriate methodology depends on the specific product attribute and its underlying mechanism of action (MoA). Generally, these potency assay formats fall into three primary categories:
- Binding assays
- Enzymatic assays
- Cell-based assays
Binding assays
Binding assays look at how well a biotherapeutic interacts with its target. For monoclonal antibodies (mAbs), receptor-based products, and related molecules, this interaction is often an important part of the mechanism of action (MoA). Depending on how the assay is set up, a binding assay can be used to compare relative binding, study binding kinetics, assess receptor blocking, or measure inhibition of a ligand-target interaction.
In this group of assays, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) are commonly used. ELISA formats are often chosen when the aim is to compare relative binding of a test sample against a reference standard. SPR is useful when more detail is needed on the interaction itself, including association and dissociation kinetics.
Binding assays are usually easier to control than cell-based potency assays and can often be run more quickly. Their limitation is that binding is only one part of the biological story. A molecule may still bind its target yet fail to produce the intended downstream biological effect. For that reason, binding assays can be highly useful, but their relevance has to be judged against the product’s MoA and the biological function the assay is expected to support.
Enzymatic assays
Enzymatic assays are useful when the biotherapeutic works through catalytic activity or operates by inhibiting a target enzyme. These assays are often clean and quantitative, and built around defined substrates, which improves method control and precision. Still, the assay must reflect the product’s key biological activity. A simple substrate system may fail to capture the biological complexity. Typical technologies for enzymatic assay read out include spectrophotometry and liquid chromatography with UV or MS detection.
Cell-based assays
Cell-based assays generally offer the highest level of biological relevance by measuring a functional response within living cells. Thus, response may include signaling cascades, proliferation, cytotoxicity, transduction, or gene expression. In practice, the readout may be captured by flow cytometry, spectrophotometry, immunoassays such as enzyme-linked immunosorbent assay (ELISA), reporter gene assays, or quantitative polymerase chain reaction (qPCR). Because they provide the strongest link to physiological function, regulators view cell-based assays as the gold standard for potency testing, in particular for later clinical phases and commercial release. However, they are inherently variable systems; cell behavior changes, reagents vary, and factors like passage number and incubation timing are critical. While. reporter gene assays can improve sensitivity and readout control; they still demand rigorous qualification and maintenance.
Practical techniques and platforms
The choice of technique depends on what the potency assay is intended to show. For binding assays, the readout is usually linked to target engagement, relative binding, or binding kinetics. For binding assays, the choice often comes down to what needs to be shown. Enzyme-linked immunosorbent assay (ELISA) is a practical option when the main question is how a test sample binds compared with a reference standard. Surface plasmon resonance (SPR) is better suited to situations where the interaction needs to be followed more closely, including how quickly the molecule associates with and dissociates from its target.
Enzymatic assays are used when product activity can be linked to a defined enzyme reaction. The signal may come from enzyme activity, substrate turnover, or the formation of a reaction product. In many cases, this is measured by spectrophotometry using chromogenic or fluorogenic substrates. If the reaction mixture contains several components, high-performance liquid chromatography (HPLC) can help separate the reaction products before they are quantified.
Cell-based potency assays usually require broader readout options because they measure a biological response in living cells. Depending on the mechanism of action, this response may be assessed by flow cytometry, spectrophotometry, immunoassays such as ELISA, reporter gene assays, or quantitative polymerase chain reaction (qPCR). These methods can be used to follow receptor activation, changes in cell-surface markers, cytokine release, transgene expression, or other functional responses linked to product activity.
A useful distinction is that the platform does not define the assay type on its own. The same technology can be used in different contexts. ELISA, for example, may be used as a direct binding assay for an antibody. The same platform may also be used in a cell-based potency assay to measure a secreted biomarker, such as interferon gamma (IFN-γ), after cellular activation. What defines the assay is the biological activity or product attribute being measured, not only the instrument or readout format.
Why potency is defined as a CQA
Potency is classified as a CQA because a biotherapeutic's clinical performance is directly dependent on its biological activity. While potency represents the functional capability of the molecule to achieve its intended therapeutic effect, the potency assay serves as the critical analytical tool used to measure and verify this activity. Maintaining a consistent potency readout across manufacturing batches, production sites, and stability timelines provides the essential data required to ensure product safety, efficacy, and lot-to-lot consistency.
Batch-to-batch consistency
Potency assay outcomes provide a direct view of batch-to-batch consistency. A drop, drift, or deviating pattern may point to process control issues, formulation changes, or a product quality problem before the issue reaches patients.
Support dose selection
Dose decisions need a link between product amount and its biological effect. Evaluation of potency helps build that link. In practice, the assay data can guide dose selection, dose adjustment, and clinical interpretation. When the product’s biological activity is inconsistent, dose decisions become harder.
Demonstrate stability over time
Stability monitoring also depends on the evaluation of potency, given that biological activity can decrease over time and storage. That drop may suggest degradation, aggregation, or other changes that impact normal product activity. A steady potency trend supports shelf life and storage conditions.
Key challenges in potency assay development
Potency assays are difficult because they measure biological activity, not only product chemistry. The closer the assay gets to the product’s actual mechanism of action (MoA), the more biological variables may emerge. This complexity peaks with advance modalities, such as viral vectors, where potency must be read alongside identity, purity, infectivity, and expression data to ensure a reliable clinical profile. For more detail, see our blog on viral vector analytical characterization.
Biological variability
One of primary hurdles of potency assay development are their inherent biological variability. Potency assays often rely on living cell lines or tissue, which are prone to environmental conditions, passage number, growth phase and reagent lot variation (media, serum). Controlled conditions and consistent workflows are essential, making that small changes should be documented and addressed before they become method noise.
Assay reproducibility
A potency assay should perform consistently across days, analysts, and labs. That is particularly challenging, given that small differences in cell seeding, incubation time, plate layout, or detection step can increase variability in the final result. Evaluating multiple assays in early development and selecting the most relevant and robust option helps to reduce this risk.
Standardization
Many potency assays lack clear industry standards. Each method is shaped by the molecule and its biology. This can complicate comparability, validation, and technology transfer across laboratories and platforms. In this context, reliable reference standards, well-defined system suitability criteria and robust assay controls are indispensable.
Analytical and statistical considerations
Potency assays need strong statistical support because they measure biological activity, not only an analytical signal. In many cases, this activity is evaluated using dose-response curves, where the relationship between concentration and response is non-linear. For this reason, a four-parameter logistic (4PL), parallel-line or slope-ratio model may be needed to describe the curve appropriately.
Once the curve is established, parallelism, curve fit, and relative potency should be assessed carefully, since these elements determine whether the test sample can be meaningfully compared with the reference standard. Replication strategy is also important. Teams should define technical replicates, biological replicates, plate layout, and repeat rules before validation begins.
System suitability criteria should be clearly defined, and acceptance limits should reflect both method capability and product risk. Together, these elements support a reliable potency assay and are closely linked to analytical method validation. For more detail, see our blog on analytical method validation.
Regulatory expectations for potency assays
Regulators understand that potency assays are inherently more variable. That is accepted when the variability is understood, managed and justified. The method must be fit for purpose, while being aligned with the product’s mechanism of action and particular stage of biotherapeutic development. A clear control strategy is essential and builds confidence towards regulators. In addition, method lifecycle management is a clear regulatory expectation; as product knowledge grows, potency methods may need to be refined, bridged, or – in the worst case – replaced.
Best practices for robust potency assay development
A robust development strategy begins with a deep understanding of the product's underlying biology. First, define the biological function most relevant to clinical efficacy, and then design the analytical method around that specific mechanism. If time and budget allows, it is considered good practice to evaluate multiple potency assay set-ups during early development, as some approaches might better capture the functional profile of the biotherapeutic than others; Because biological systems require strict control, careful management must be applied to cell lines, critical reagents, reference standards, and data analysis.
Implementing statistical process control to monitor assay performance over time is essential for identifying potential failures early and detecting shifts before they impact product timelines. Partnering with an experienced Contract Research Organization (CRO) can streamline this process; their specialized expertise supports assay design, troubleshooting, qualification, and validation. For expert, phase-appropriate analytical support, explore Anacura.
Connect with our experts
Our experts support pharmaceutical and biotech companies in developing and validating potency assays that accurately reflect the biological activity of biotherapeutics throughout the product lifecycle.
From assay selection and method development to validation, statistical evaluation, and regulatory alignment, we help ensure reliable potency testing that supports product quality, consistency, and compliance.
Get in touch to discuss your program and explore how we can support your analytical strategy.
Frequently asked questions
What is a potency assay for biotherapeutics?
A potency assay measures biological activity of the therapeutic. It confirms that the product carries out its intended biological function.
Why are cell-based assays preferred?
Because cell-based assays offer the highest level of biological relevance and provide the strongest connection to physiological function, they are preferred for potency testing, especially at later clinical stages and commercial release.
What are the main challenges in potency assay development?
Three problems usually cause the most friction: biological variability, reproducibility, and standardization.
How do regulators view variability in potency assays?
Regulators recognize that potency assays are inherently more variable Variability can be accepted when justified, managed and controlled by means of appropriate system suitability control and defined acceptance limits.
What is the difference between binding assays and functional potency assays?
Binding assays tell you whether a molecule binds to its target. Functional assays tell you whether that binding actually causes a downstream biological effect.
