INTRODUCTION
Early potency assurance offers several benefits including: (1) reducing the risk of late-stage failures, (2) improved product quality and consistency, and (3) accelerated development timelines. Typically, potency assays are developed and qualified or validated at the end of the manufacturing cycle prior to entering clinical trials. However, the potency assay should be implemented at various stages of manufacturing. Establishing robust potency assays early in manufacturing, using a research or engineering grade of drug, allows for process optimization to consistently deliver GMP products within defined potency criteria and minimizes failures. Well-characterized potency assays employing GMP products provide clear, dependable data packages with the core requirements for FDA submissions, facilitating smoother, quicker review and approval processes.
A well-designed potency assurance strategy is essential for the successful progression of pharmaceuticals from discovery through commercialization. This strategy serves as a systematic approach to ensuring that each manufactured product lot consistently meets its intended therapeutic effect. Central to this is the application of quality risk management, which involves identifying, evaluating, and controlling potential risks to potency throughout the product lifecycle.
The development of a potency assurance strategy begins with the application of prior knowledge and experience, leveraging historical data and scientific understanding to inform initial decisions. As product and process understanding deepens—through both preclinical and clinical development—this knowledge base is expanded, enabling more precise control and assurance of potency. Risk assessment is a critical component, requiring the identification of potency-related critical quality attributes (CQAs) early in development. These CQAs are key risk properties that must be maintained to ensure product efficacy and safety. Critical Quality Attributes (CQAs) are the physical, chemical, biological, or microbiological properties or characteristics that must be controlled to ensure the quality, safety, and efficacy. In the context of cell-based potency assays, CQAs are specifically those attributes that relate directly to the functional biological activity (potency) and overall performance of a product. The potency assurance strategy will cite CQAs during the manufacture process, the materials for manufacture and assay, product-contact containers, closures, in-process testing, and the potency assay for lot release. By systematically assessing risks to these attributes, manufacturers can design robust manufacturing processes, implement appropriate material controls, and establish in-process testing protocols. This proactive approach minimizes the likelihood of failed product lots and supports consistent product quality.
As additional manufacturing and clinical data are gathered, the potency assurance strategy should be continuously refined. This includes updating control strategies, potency assays, and acceptance criteria to ensure they remain phase-appropriate and effective at rejecting lots that are either sub-potent or have excess activity. All changes and justifications must be thoroughly documented in regulatory submissions, demonstrating a commitment to ongoing quality risk management and regulatory compliance.
Assay selection and design are foundational steps that require careful consideration of the product’s mechanism of action and intended clinical effect. The chosen assay must possess desirable characteristics such as specificity, sensitivity, accuracy, and reproducibility, among other criteria. Approaches to assay selection and design should be scientifically justified, leveraging both prior knowledge and empirical data to ensure the assay is fit-for-purpose.
Once a suitable potency assay is identified, its qualification and validation are essential to demonstrate its reliability and robustness in measuring potency. Qualification establishes that the assay performs as intended under defined conditions, while validation provides formal evidence of its suitability for routine use. Both processes must adhere to regulatory expectations and industry best practices.
Acceptance criteria define the quantitative thresholds that a product lot must meet to be considered potent and suitable for release. These criteria are established based on manufacturing, preclinical and clinical data, and are refined as additional information becomes available. Acceptance criteria must be scientifically justified, phase-appropriate, and aligned with regulatory requirements to ensure patient safety and product efficacy.
Ultimately, the integration of well-designed potency assays and clearly defined acceptance criteria into the overall potency assurance strategy is vital. This ensures that each product lot released to the market meets stringent quality standards and delivers the intended therapeutic benefit to patients.
DEVELOPING A POTENCY ASSURANCE STRATEGY
Product quality and therapeutic consistency requires tightly controlled and documented assay attributes. This process begins with a thorough understanding of the product’s mechanism of action (MOA), its clinical indication, and the route of drug administration. It also requires establishing manufacturing process controls and defining critical quality attributes (CQAs) to consistently produce drug lots that meet predetermined specifications. While prior knowledge and experience—such as data from nonclinical studies, literature, or characterization studies—can inform the initial design of a potency assurance strategy, manufacturers must recognize that differences in products, including mechanism of action, (MOA) intended therapeutic effect, manufacturing processes, and starting materials, can impact potency in unforeseen ways. For example, in autologous cell therapy products (obtained from the same individual) the potency may be influenced by the disease state or treatment history of the donor cells. Therefore, it is vital to conduct product- and process-specific characterization studies and risk assessments, rather than relying solely on prior knowledge, to ensure a comprehensive and effective strategy.
Product characterization studies conducted from the earliest research phases help elucidate the MOA and identify key product attributes, which in turn better define the relevant CQAs as they relate to potency. However, if the MOA is not unequivocally defined, then the relationship between the clinical outcome and the product’s attributes may suggest relevancy to potency. The potency assay for lot release should utilize a functional assay pertinent to the clinical outcome, which may not be the MOA.
Supplier qualification is also a critical component—not only during manufacturing but also during the actual potency assay. Vendor and supplier risks must be carefully managed, as they can significantly impact CQAs and, consequently, product potency. All manufacturing processes that affect potency should be subject to rigorous risk assessment and mitigation strategies. Furthermore, if the MOA is not unequivocally defined, the relationship between clinical outcomes and product attributes may provide important insights into potency relevance. Using unqualified or inconsistent assays introduces uncertainty and may fail to detect changes in product potency that could affect clinical relevance. Therefore, robust assay qualification and ongoing risk management are essential to maintain the integrity and reliability of the potency assurance strategy.
POTENCY ASSAYS
The Role of CQAs and Potency Assurance Strategies when Developing Potency Assays
Developing a reliable assay to measure potency is crucial for confirming the drug’s intended therapeutic effect and for meeting regulatory requirements. Potency assurance strategies are equally important, as they ensure that the potency assay is validated and robust. This includes demonstrating the assay’s accuracy, precision, specificity, and reliability, as well as establishing controls to monitor and maintain potency throughout manufacturing and the product lifecycle. These strategies help manage risks by identifying and controlling sources of variability that could impact potency.
Uses of Potency Assays
Potency assays play a crucial role in drug development by ensuring that a product maintains its intended biological activity throughout its lifecycle. These assays should be robust enough to detect loss of activity, including through forced degradation studies. Stability testing, both long-term and accelerated, should also be performed to assess potency over time, with results reflected in the product’s CQAs. One primary application is lot release testing, which confirms consistent potency between manufactured batches. Additionally, potency must be evaluated across all relevant delivery systems and use conditions, including worst-case scenarios during preparation and administration.
Potency assays should be integrated throughout the design and implementation of the manufacturing process, including during product characterization, qualification, and ongoing process verification. If manufacturing changes occur, comparability studies are essential to ensure that potency and clinical performance remain unaffected. These studies help maintain consistent CQAs and reduce risk to therapeutic outcomes. When biological activity must be measured, at least one bioassay should be used. While the assay doesn’t need to replicate the product’s mechanism of action (MOA), a solid understanding of the MOA is essential for selecting appropriate CQAs.
Desirable Characteristics of Potency Assays
Identifying the appropriate cell-based potency assay is as important as the manufacture of the drug. A manufactured lot cannot be released until it passes the required criteria for potency. The assay needs to not only evaluate the drug action but also the safety in early clinical studies. It may not unequivocally identify the MOA, but should reflect the function of the drug, so that if there are problems with manufacture or stability of the drug, the assay results will show that there are issues. Not only does the assay need to reflect the potency and efficacy of the drug, but the FDA requirements will have to be met for any approval of clinical studies. The importance of the appropriate potency assay goes hand in hand with the manufacture of the drug with the desired therapeutic effect.
Precision, accuracy and specificity are desirable characteristics of potency assays. Precision refers to the degree of agreement or consistency among repeated measurements of the same sample under specified conditions. It is a key parameter in assay validation, ensuring that the assay produces reproducible results when the same procedure is applied multiple times to the same homogeneous sample. Potency assay precision can be typically accomplished by measuring product potency against a reference standard material. However, if the assay precision cannot be achieved, then performing multiple independent assay runs for each sample and reporting the mean value must be done. Accuracy in potency assays is a measure of how close the assay’s results are to the true biological activity of the drug, as established by a reference standard or accepted value. This is essential for ensuring that the assay provides reliable data for drug development and regulatory approval. The accuracy of the product data should match the expected values over the defined range of the assay. Specificity refers to the assay’s ability to measure a unique response to the intended analyte (the drug or its active component) in the presence of potentially interfering substances. The specificity of the assay should demonstrate that non-potent product, denatured product, manufacture impurities or sample matrices have no activity in the bioassay.
A robust potency assay is crucial in drug development for clinical use because it directly ensures a drug’s safety, efficacy, and consistency throughout its lifecycle. Without a robust potency assay, drug developers face significant risks: inconsistent product quality, regulatory delays or rejection, potential safety issues, and ultimately, failure to deliver effective therapies to patients. If the assay relies on multiple steps, but the final outcome of potency is dependent upon the last step, then the assay is sufficient to demonstrate the earlier steps are active. For example, if a later step in the chain of biological activities is completely dependent on the earlier steps, then a bioassay at the later step that adequately ensures the product’s biological activity at that step will typically be sufficient to also ensure the biological activities at the earlier steps.
Assay Selection & Design
Selection and design of the appropriate assay are required for successful results. The selection and design of the assay are dependent on the drug modality. There are many types of drugs, ranging from the traditional small molecules, large molecules such as proteins and antibodies, nucleic acid derived (such as DNA or mRNA delivery) and cell therapies. Several categories are important for manufacture as well as for potency assay development. These are the in-process parameters, such as culture time, passage number, items that affect the cellular performance and output. In-process testing for cell-based potency assays include characteristics such as cellular growth rate, viability, phenotype. Lot release testing involves setting criteria that would affect the potency assay, such as freezing of culture supernatants.
The CQAs and their selection rationale must be included in regulatory submissions, with ongoing refinement as manufacturing and clinical data accumulate. The implementation process requires clear documentation of how CQAs are identified and managed, details of potency testing or alternative controls, and plans for further characterization and continuous improvement.
Should one facet of the Potency Assurance Strategy not be controlled, then to decrease the overall risk, the other facets of this strategy should be strengthened. For example, if the lot-release potency assay is not robust, or a half-life of a drug is too short, then other aspects of the potency assurance strategy should demonstrate greater stringency. As the product moves through clinical trials, additional experience and data with manufacturing process control and strategy will improve the potency assurance. This is important when applying for further clinical testing.
CQAs and Potency Assurance Strategies for Complex Drugs
It is important to recognize that there may be complications in testing the drug due to its complexity.
For complex products such as cells or genetic therapies, not only is biological activity testing necessary but physiochemical assays are considered necessary. The biological activity may not mimic the products MOA, but the MOA should guide the CQA selection. The underlying purpose of the bioassay is to quantitate the risk of potency-related CQA. For multiple active ingredients, separate testing of ingredient’s strengths are required unless the biological activity measurement measures the combined activity of the product. Often the bioactivity needs the combination of API ingredients, whereupon it should be sufficient to not have separate API assays. Conversation with the FDA with submittal of bioactivity data needs to be discussed. If CQA risks cannot be ameliorated, then the CQA should include the product’s lot release specification.
A drug delivery system will be an additional variable in the selection of the correct potency assay. For example in gene delivery, virus moieties contain DNA or RNA as the active pharmaceutical ingredient (API). Viral packaging is necessary to deliver nucleic acids to the target cell. A cellular response to the gene being delivered is required in order to develop a functional assay. Antibody drug conjugates (ADCs) consist of a target specific monoclonal antibody conjugated to a cytotoxic drug. Therefore, two distinct mechanisms that make the drug effective must be considered: the antibody that targets a cell of interest (the delivery) and the cytotoxic drug (the payload). Nanoparticles deliver drugs, whether it be small molecules, proteins or other types of drugs. The functionality of certain drugs cannot be tested without this delivery system. Exosomes, like liposomes, is another system that delivers drugs that may not normally enter cells without their bilaminar membrane. As such, when selecting a cell-based potency assay for complex drugs, it is extremely important to consider the individual components and how the drug has to be tested as a unit. Testing individual components of a complex drug may require specific assays, such as an ELISA, or binding. Assays such as these may be used to test the delivery component, or the API.
To adequately test the functionality of the drug, a cell base assay is necessary, and should reflect the action of the drug with its delivery system. The selection of the appropriate cell-based assay for complex drugs should measure the potency of the product. For example, viruses, even though their API may be a nucleic acid, such as DNA or RNA, the assay must take into account the mechanisms required for viral entry as well as the cellular response to the nucleic acid being delivered. The assay must measure the combined activity of the virus and nucleic acid to determine potency, such as expression of the transgene being delivered by the virus. The potency of the virus/nucleic acid complex is then used for clinical or pre-clinical studies. The potency assurance strategy for testing the virus-nucleic acid therapeutic moiety must take into account the CQA of the vector and the CQA of the nucleic acid. Risk assessment of the nucleic acid could be the sequence, length or binding activity. If the risks cannot be mitigated, then additional potency assays with their quantitative acceptance criteria will be necessary.
Another example of a complex drug is a patient-specific cellular product which is transduced with a vector such as a CAR T cell. The lot release should include acceptance criteria for both the cellular product and for the vector used for the transduction. For the tissue-engineered product, which may require scaffolds, cells, and culture specific technology, the CQAs should include properties such as biomolecular, biochemical, immunological or other components pertinent to the modified cell/tissue. If the CQA cannot fully mediate the risk assessment, then CQA should be included in the product’s lot release acceptance criteria.
A common factor between developing potency assays of a complex drug and drug-delivery system, is cell selection. The cell is the most variable component of any potency assay. The assay selection should relate to the biological activity of the drug or drug-delivery system as it would relate to an in vivo situation targeting appropriate tissues. For the sake of this white paper, the API combined with the drug-delivery system will be referred to as the drug.
Selection of an assay is not only selection of the correct “target” cell type but the read out. For instance, a drug that activates a T cell to synthesize and release TNF-a, will require a cell that is either a T cell or a cell line that mimics a T cell, and is able to express TNF-a upon activation.
For complex products, which often have multiple potency-related CQAs that cannot be adequately controlled without release testing. A potency assurance strategy should generally include several release assays, each quantifying a potency-related CQA at risk. These may involve physicochemical assays and/or bioassays. Some potency-related CQAs linked to biological activity can only be effectively measured with a bioassay; in such cases, at least one bioassay should be included, focusing on quantifying a relevant CQA rather than mimicking the product’s mechanism of action (MOA). For complex products with multiple active ingredients, lot release testing must assess the strength of each ingredient, requiring measurement of their concentration or potency. In some cases, a single bioassay may suffice for all active ingredients, supplemented by physicochemical assays to measure each ingredient’s concentration. Risk assessment and prior knowledge should guide assay design and control strategies to ensure reliable assay performance and mitigate unacceptable risks.
Assay Control and Change Management
In drug development, assay acceptance criteria are predefined standards or ranges that must be met for an assay to be considered valid and for its results to be accepted. These criteria ensure that both the analytical system and the samples being tested are suitable for generating reliable and interpretable data. System suitability refers to checks performed to confirm that the assay system (including equipment, reagents, and procedures) is functioning correctly before or during sample analysis. The purpose is to ensure that the analytical method can deliver accurate and reproducible results. Sample suitability assesses whether each individual test sample is appropriate for analysis and if its results can be reliably interpreted. Whereas system suitability ensures the overall assay system is functioning properly, sample suitability ensures each individual sample result is reliable and interpretable. Both are essential for regulatory compliance and for ensuring the integrity of data used in drug development and decision-making. To determine if an assay is not reliable, the pre-defined assay acceptance criteria for sample suitability and system suitability should be established. However, depending upon the stage of the clinical trial, some companies will report sample suitability when enough data is available, however, system suitability will denote that the assay was functional. The system suitability will employ reference material as well as positive controls and negative controls to demonstrate that the assay is performing as expected. The data acquired over time will allow identification of any adverse trends in potency.
The reference material which is used as a basis of the sample behavior in the assay, can be a single lot of drug product. The reference material should be qualified initially and tested during ongoing stability studies. If the reference material is replaced, the new material must undergo the same statistical qualification in comparison to the original reference material. The same is true for assay controls. These qualifications typically use multiple potency assay experiments, resulting in an established and expected potency range for reliability and accuracy of the assay system.
Assay Qualification and Validation
Some key performance indicators of the assay system are: accuracy, precision, specificity and sensitivity. Qualification is “fit-for-purpose” for early-stage clinical studies with validation needed for later stage clinical studies. The CQAs for risk assessment will identify the parameters that need to be controlled in order for the assay to be used to assure product potency. For drug product to be released for a licensed product, the assay must be validated. During validation, a complete understanding of the risks for each step of the drug life should be completed. This will also include a complete understanding of the potency release test, with the risks associated with the assay. To proactively identify the potential sources of variability that could compromise the assay performance, could be differences in instrumentation, analysts, lots of biological reagents and cells. To mitigate this, some of the risks could be modified by using various supply vendors, different lots of reagents, and different methods for culturing cells. If the potency assay was found to be deficient, and a change in the assay is warranted, the new assay should be performed in an equivalence test against the original assay. If the results are similar to each other, it will ensure consistency and reliability.
Acceptance Criteria
Quantitative acceptance criteria are necessary to ensure the CQAs are addressed as risks to assure product potency. The risk management approach should be used and the CQAs refined with increasing assays that are performed. For a potency assay, a lower and upper quantitative limit is defined. The lower limit ensures that the product can be detected accurately and that it will have the appropriate therapeutic activity. The upper limit defines that the drug will not be in an unsafe safety range. For the upper quantitative limit, using manufacturing data, non-clinical studies and clinical data will ensure that the drug will remain in the safe potency range. For cellular products, the range of acceptance will be larger than small molecules because of the inherent variability of cells. However, the range should ensure that that if the drug tests outside of the acceptance criteria, that the lot of drug will be rejected. For licensed products, the potency release acceptance criteria must be related to the effectiveness of the product. It should be accurate and robust enough to ensure that the distribution lots match the lots used for the clinical trials, providing consistency and reliability to patients. The broader use of potency assays is the tracking of stability studies, compatibility with delivery devices and assurance of comparability after manufacturing changes .
CONCLUSION
Establishing a robust potency assurance strategy and identifying potency-related critical quality attributes (CQAs) early in drug development are essential for ensuring product quality, safety, and efficacy. Early selection and justification of CQAs—such as in-process parameters, cellular characteristics, and lot release criteria—provide a strong foundation for both manufacturing consistency and regulatory compliance. Clear documentation of how CQAs are identified, managed, and refined, alongside detailed potency testing strategies, is vital for regulatory submissions and continuous improvement as clinical and manufacturing experience grows.
For complex products, such as cell or gene therapies, a comprehensive approach that integrates both biological activity and physicochemical assays is necessary. The mechanism of action (MOA) should guide CQA selection and the functional assay (if the MOA is unknown), and for products with multiple active ingredients, testing strategies must be tailored to accurately reflect the product’s combined potency. Ongoing dialogue with regulatory authorities, such as the FDA, is crucial to ensure that bioactivity data and CQA management meet evolving expectations.
Ultimately, if any aspect of the potency assurance strategy cannot be fully controlled, compensatory measures should be implemented to mitigate overall risk. As products advance through clinical development, continuous refinement of potency assurance strategies and CQAs will support successful regulatory approval and, most importantly, patient safety.
FOR FURTHER READING
Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (US FDA)
RELATED ARTICLES / BLOGS
- Developing Robust Potency, Pharmacokinetics (PK), and Pharmacodynamics (PD) Assays for Antisense Oligonucleotide (ASO) Therapeutics
- Developing and Validating a Potency Assay for Oncolytic Viruses: Insights from the Coxsackievirus A21 Study https://www.marinbio.com/developing-and-validating-a-potency-assay-for-oncolytic-viruses-insights-from-the-coxsackievirus-a21-study/
- Organoids in the Development of Pharmacokinetics, Pharmacodynamics, and Potency Assays https://www.marinbio.com/development-of-pharmacokinetics-pk-pharmacodynamics-pd-and-potency-assays-with-organoids/
- Development and Validation of Cell-Based Potency Assays for AAV Therapies: Insights From a Commercial AAV Vector Study https://www.marinbio.com/development-and-validation-of-cell-based-potency-assays-for-aav-therapies-insights-from-a-commercial-aav-vector-study/
- Precision in Efficacy: Developing Robust GMP Potency Assays for PROTACs, Molecular Glues, and Other Targeted Protein Degradation (TPD) Drugs https://www.marinbio.com/precision-in-efficacy-developing-robust-gmp-potency-assays-for-protacs-molecular-glues-and-other-targeted-protein-degradation-tpd-drugs/
- Advancing Cancer Therapeutics: The Role of Potency Assays in ADC Effectiveness
- https://www.marinbio.com/revolutionizing-cancer-treatment-how-potency-assays-make-adcs-more-effective/
- Comprehensive Guide to Potency Assay for Drug Lot Release https://www.marinbio.com/drug-lot-release-potency-assay-testing/
