Navigating EMA vs FDA Cell Therapy Potency Testing: A Strategic Guide for Global Development

Abstract

This article provides a comprehensive comparison of the European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) requirements for cell therapy potency testing. Aimed at researchers, scientists, and drug development professionals, it explores the foundational regulatory frameworks, details methodological approaches for assay development, offers strategies for troubleshooting common challenges, and guides the validation and comparability process. By synthesizing current guidelines and real-world case studies, this guide aims to equip developers with the knowledge to navigate divergent regulatory expectations, optimize their testing strategies, and accelerate global market entry for innovative cell-based therapies.

Understanding the Regulatory Landscape: EMA and FDA Foundational Principles for Potency

In the realm of biological medicines, particularly for cell and gene therapy products (CGTs), potency stands as a defining Critical Quality Attribute (CQA) that separates these complex products from conventional small-molecule drugs. According to regulatory definitions, potency is "the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result" [1]. Unlike small-molecule drugs where potency often directly correlates with active ingredient concentration, potency in biological products depends on multiple interacting factors that collectively determine the overall therapeutic effect [2]. This fundamental distinction necessitates sophisticated testing approaches that can capture the biological activity reflective of the product's intended clinical performance.

The designation of potency as a CQA means it represents a property that must remain within specified limits to ensure the safety and efficacy of the product [2]. For cell and gene therapies, also classified as Advanced Therapy Medicinal Products (ATMPs), potency testing presents unique challenges due to their complex nature—they may consist of viable cells, viral vectors, or nucleic acids with intricate mechanisms of action [1]. The lot release process specifically relies on robust potency measurements to ensure that each manufactured batch possesses the specific biological activity required to achieve the intended therapeutic effect in patients, thereby guaranteeing consistency and reliability across production lots [3] [4].

Regulatory Framework: EMA vs. FDA Requirements

Comparative Analysis of Regulatory Expectations

The regulatory landscape for potency testing of cell and gene therapies demonstrates both convergence and divergence between the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA). Both agencies recognize potency as a critical quality attribute that must be measured for lot release, yet they exhibit differences in their specific requirements and implementation timelines [5] [1].

The table below summarizes key distinctions between EMA and FDA requirements based on current guidelines:

Regulatory Aspect	FDA Requirements	EMA Requirements
Primary Guidance Documents	"Potency Assurance for Cellular and Gene Therapy Products" (2023 draft) [6]; "Potency Tests for Cellular and Gene Therapy Products" (2011) [7]	Guideline on quality of ATMPs (2025); Human cell-based medicinal products guideline; Gene therapy guidelines [5] [1]
Method Validation Timing	Typically not required for Phase 1 INDs [5]	"An appropriate degree of method validation should be applied at each stage"; safety-relevant assays must be fully validated [5]
Assay Type for Release	Expects a quantitative functional potency assay for release [1]	May allow validated surrogate assays for release when correlated with functional characterization assays [1]
Early Development Approach	Recommends evaluating multiple potency assays during early development [1]	Similarly recommends multiple assay evaluation during early development phases [1]
Legal Basis	21 CFR Part 600.3 [1]	EU Directive 2001/83/EC, Annex I, Section 3.2.2.1 [1]

A significant challenge for developers emerges from inconsistent phase-dependency of regulatory expectations between agencies. The FDA's 2020 guidance for Chemistry, Manufacturing, and Control (CMC) information in Investigational New Drug Applications (INDs) specifically indicates that validation of analytical methods is typically not required for Phase 1 submissions. In contrast, the 2025 EMA guideline on the quality of ATMPs states that "an appropriate degree of method validation should be applied at each stage" and mandates that all safety-relevant assays must be fully validated [5]. This misalignment creates complexity for sponsors developing global development programs.

Analysis of Approved Products and Testing Strategies

A comprehensive review of the 31 FDA-approved cell therapy products through 2024 reveals insightful patterns in potency testing strategies adopted by successful programs. On average, each approved product utilized 3.4 potency tests (standard deviation 2.0), with a median of 3.0 tests per product [3]. The distribution ranged from products with only a single potency test to one product (Lenmeldy) that reported eight distinct potency tests [3].

The following table categorizes the 71 non-redacted potency tests used by these approved products, illustrating the prevalence of different testing approaches:

Potency Test Category	Number of Tests	Percentage	Example Applications
Viability and Count	37	52%	Cell viability measurements; viable cell counts [3]
Expression	19	27%	CAR expression from viable T cells; protein expression markers [3]
Bioassays	7	7%	Functional activity measurements; cytotoxicity assays [3]
Genetic Modification	6	9%	Vector copy number; transgene expression analysis [3]
Histology	2	3%	Tissue structure and composition assessment [3]

Notably, 61% of approved CTPs used measurements of cell viability or cell count as a potency test, while 65% utilized gene or protein expression measurements [3]. The most frequent combination consisted of "Viability and count" together with "Expression" tests, which occurred for 16 CTPs (52%) [3]. This data suggests that while regulators emphasize the importance of functional bioassays, many approved products successfully navigate the regulatory process using a combination of physical, phenotypic, and molecular measurements as part of their potency testing matrix.

Potency Assurance Strategy and Testing Methodologies

Developing a Comprehensive Potency Assurance Strategy

Modern potency assurance extends beyond a single lot-release test to encompass a multifaceted strategy that reduces risks to product potency through manufacturing process design, process controls, material controls, in-process testing, and final product potency assays [6]. This comprehensive approach ensures that every product lot released will have the specific ability or capacity to achieve the intended therapeutic effect [6]. The foundation of this strategy rests on a deep understanding of the product's mechanism of action (MoA), which should directly inform the design of potency assays [5] [4].

The BioPhorum ATMP working group has addressed the challenges of developing integrated potency strategies by creating a Gene Therapy Potency Strategy Roadmap—an interactive aid designed to help developers navigate complex decisions and propose simplifications for an ideal future state [5]. This initiative acknowledges the persistent ambiguity around strategic questions such as the advantages of various methods, phase-appropriateness of testing strategies, the sufficiency of surrogate method baskets, and whether the overall strategy adequately interrogates the intended MoA [5]. The roadmap emphasizes embedding potency strategy early in development planning to create a foundation for alignment and uniformity of regulatory expectations.

Diagram: Comprehensive potency assurance strategy integrates multiple elements from early development to regulatory compliance.

Methodological Approaches to Potency Testing

In Vitro vs. In Vivo Potency Assays

The evolution of potency testing methodologies has witnessed a significant shift from traditional in vivo assays to more controlled in vitro systems. In vivo assays, which involve administering the product to animal models and measuring physiological responses, face substantial challenges including ethical concerns, resource intensity, lengthy trial periods, and high variability—with studies showing %CV of in vivo potency measurements ranging from 34% to 125% [2]. In contrast, modern in vitro methods demonstrate significantly lower variability, with %GCV often below 10% for similar vaccines, while also offering faster turnaround times and reduced ethical concerns [2].

For gene therapy products, particularly those based on adeno-associated virus (AAV), in vitro potency assays typically involve transducing permissive cell lines with the vector and measuring downstream biological responses [4]. These assays are designed to capture how cells biologically respond to the vector, with measurements potentially including vector-derived RNA or protein quantification, enzymatic activity, changes in gene expression patterns, or more complex phenotypic changes [4]. The selection of an appropriate cell line represents a critical consideration, as it must demonstrate compatibility with the vector's tropism, promoters, and the therapy's mechanism of action [4].

Analytical Methods and Statistical Approaches

Robust potency testing employs various analytical methods tailored to product characteristics, with three primary statistical models commonly used to analyze potency assay data [4]. The parallel-logistic analysis method utilizes a three-, four-, or five-parameter logistic regression model to generate a dose-response curve for precise calculation of relative potency. The parallel-line analysis approach employs linear regression to evaluate relative potency based on parallel dose-response relationships, while the slope-ratio analysis also uses linear regression models with different mathematical treatment [4].

The concept of relative potency represents a cornerstone of potency testing, where test samples are compared against a reference standard of known properties in the same experiment [2]. This approach accounts for experimental variability and enables more precise results by normalizing for day-to-day and operator-to-operator variations. The implementation of well-characterized reference standards is particularly important for cell-based assays, which may exhibit higher intrinsic variability than biochemical methods [1].

Experimental Design and Research Toolkit

Potency Assay Development Workflow

The development of a robust potency assay follows a structured lifecycle encompassing development, qualification, and validation phases [4]. During the development phase, critical parameters including cell line selection, transduction conditions, and experimental readouts are optimized. This phase requires careful consideration of the therapy's mechanism of action to ensure the assay captures biologically relevant activity [4]. The qualification phase demonstrates that the method is suitable for its intended purpose, while validation—required for commercial release testing—establishes formal performance characteristics including precision, accuracy, specificity, and robustness [1] [4].

Diagram: Potency assay development progresses through defined stages from early development to commercial validation.

The Scientist's Toolkit: Essential Research Reagents and Materials

The implementation of robust potency testing requires carefully selected reagents and materials designed to ensure reproducibility and regulatory compliance. The following table details essential components of the potency testing toolkit:

Tool/Reagent	Function	Application Notes
Mechanism of Action-Relevant Cell Line	Provides biological system for assessing functional activity	Must demonstrate compatibility with vector tropism and promoters; require proper cell banking and testing [4]
Reference Standard	Serves as comparator for relative potency calculations	Well-characterized material stored under controlled conditions; essential for assay standardization [2]
Vector-Specific Detection Reagents	Enable quantification of transgene expression	Includes antibodies for protein detection, primers/probes for nucleic acid detection [4]
Cell Culture Media & Supplements	Support cell growth and maintenance	Serum-free formulations preferred for consistency; require qualification testing [4]
Validation Samples	Establish assay performance characteristics	Include samples representing expected specification ranges [1]

A critical decision in assay development involves selecting between mechanism of action-reflective assays using tissue-relevant cell lines versus more practical surrogate assays [5]. While MoA-reflective assays represent the gold standard for GMP release and stability testing, they must be reviewed from a quality control perspective to ensure they are sufficiently robust and reproducible for the stringent GMP operating environment [5]. Early development cell lines used for target selection may have use restrictions that must be navigated prior to implementation in clinical and commercial supporting assays [5].

For products with complex or unknown mechanisms of action, a basket of surrogate assays may be implemented, particularly when these assays demonstrate greater amenability to validation and performance in quality control environments [5]. These surrogate assays may not be cell-based and could instead employ physiochemical methods if the measured property correlates with therapeutic activity relevant to the MoA [5]. A well-supported basket of surrogate assays can dovetail with existing potency methods to provide an overall potency strategy that is both rigorous and well-controlled.

The definition of potency as a critical quality attribute and its central role in lot release represents a cornerstone of cell and gene therapy development and regulation. The complex nature of these advanced therapies necessitates sophisticated testing strategies that capture biological activity reflective of the intended therapeutic effect. While regulatory agencies universally recognize the importance of potency testing, divergent expectations between the EMA and FDA regarding method validation timelines and acceptable assay types for release create challenges for global development programs.

The analysis of successfully approved products reveals that most employ a multi-attribute approach to potency testing, combining viability measurements, phenotypic characterization, and increasingly, functional bioassays. As the field continues to evolve, the development of standardized potency roadmaps and continued regulatory dialogue will be essential to harmonize expectations while maintaining the flexibility needed to address the unique characteristics of individual products. Through continued scientific advancement and regulatory collaboration, the field moves toward more meaningful, robust, and efficient potency testing strategies that ultimately benefit patients awaiting these transformative therapies.

The Center for Biologics Evaluation and Research (CBER) within the U.S. Food and Drug Administration (FDA) regulates cellular and gene therapy (CGT) products as biological products under Section 351 of the Public Health Service Act [8]. The Office of Therapeutic Products (OTP) in CBER is specifically responsible for reviewing these innovative therapies [9]. For drug development professionals targeting both U.S. and European markets, understanding the distinct regulatory philosophies of the FDA and the European Medicines Agency (EMA) is crucial. A fundamental difference lies in their approval authority: the FDA has full approval authority for CGTs, whereas the EMA provides a scientific opinion with the final decision made by the European Commission [9]. This guide provides a detailed comparison of FDA and EMA requirements, focusing on potency testing, with supporting experimental data and protocols to inform global development strategies.

The FDA has established a comprehensive framework of guidances to navigate the complex development pathway for CGT products. These documents reflect the agency's effort to provide clarity while addressing the unique challenges of these innovative therapies.

Foundational and Recent Potency Guidances

Potency Tests for Cellular and Gene Therapy Products (January 2011): This final guidance provides the foundational recommendations for developing tests to measure potency for Investigational New Drug Applications (INDs) and Biologics License Applications (BLAs) [7]. It clarifies that because potency measurements are designed for a particular product, it does not recommend specific types of potency assays nor propose acceptance criteria for product release [7].

Potency Assurance for Cellular and Gene Therapy Products (Draft, December 2023): This draft guidance represents a significant evolution in regulatory thinking, proposing a comprehensive, risk-based approach to potency assurance throughout the product lifecycle [8]. When finalized, it will supersede the 2011 potency guidance [8]. It recommends a multifaceted "potency assurance strategy" that reduces risks to potency through (1) manufacturing process design, (2) manufacturing process control, (3) material control, (4) in-process testing, and (5) potency lot release assays [8]. The American Society of Gene & Cell Therapy (ASGCT) has emphasized the need for this final guidance to address phase-appropriateness throughout, noting that robust potency assays critical in late-stage development may not be feasible early in clinical development due to assay complexity, small sample sizes, and incomplete mechanistic understanding [10].

Additional Key CBER Guidances

The FDA has issued numerous other guidances addressing various aspects of CGT development:

• Human Gene Therapy Products Incorporating Human Genome Editing (January 2024) [11]
• Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products (January 2024) [11]
• Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products (Draft, July 2023) [11] [12]
• Innovative Designs for Clinical Trials of Cellular and Gene Therapy Products in Small Populations (Draft, September 2025) [11] [13]
• Long Term Follow-up After Administration of Human Gene Therapy Products (January 2020) [11]

Comparative Analysis of FDA and EMA Regulatory Frameworks

Navigating the divergent regulatory expectations between the FDA and EMA presents significant challenges for CGT developers. A recent study published in JAMA Internal Medicine found that only 20% of clinical trial data submitted to both agencies matched, revealing major inconsistencies in regulatory expectations [9].

Key Regulatory Differences: FDA vs. EMA

Table 1: Comparison of FDA and EMA Regulatory Processes for CGT Products

Aspect	FDA (U.S.)	EMA (EU)
Clinical Trial Approval	IND Application; 30-day review before trials can begin [9]	CTA submitted to National Competent Authorities and Ethics Committees; centralized submission via CTIS possible [9]
Marketing Approval	Biologics License Application (BLA) [9]	Marketing Authorization Application (MAA) [9]
Review Timelines	Standard: 10 months; Priority: 6 months [9]	Standard: 210 days; Accelerated: 150 days [9]
Regulatory Classification	RMAT designation for expedited review [9]	Classified as Advanced Therapy Medicinal Products (ATMPs) [9]
Long-Term Follow-Up	Requires 15+ years for gene therapies [9]	Risk-based, generally shorter than FDA [9]
Expedited Pathways	RMAT, Fast Track, Breakthrough Therapy, Accelerated Approval [9]	PRIME Scheme, Conditional Marketing Authorization, Accelerated Assessment [9]
Decision-Making Authority	FDA/CBER has full approval authority [9]	EMA provides scientific opinion; European Commission makes final decision [9]

Strategic Implications for Global Development

The regulatory divergence between FDA and EMA creates substantive challenges for CGT developers:

• Data Inconsistencies and Increased Costs: Sponsors must prepare distinct applications for each agency, adapting trial protocols and evidence to meet differing expectations [9].
• Approval Timeline Variance: The FDA often accepts real-world evidence and surrogate endpoints, potentially enabling faster market access, while the EMA typically requires more comprehensive clinical data, emphasizing larger patient populations and long-term efficacy [9].
• Manufacturing and Control Differences: Critical manufacturing differences exist in areas such as starting materials definition, viral vector classification, and potency verification requirements [12].

Potency Testing Requirements: FDA vs. EMA Comparative Analysis

Potency testing represents one of the most challenging aspects of CGT development, with FDA and EMA exhibiting both convergent and divergent expectations.

Analysis of Potency Tests from FDA-Approved Products

A 2025 analysis of the 31 U.S. FDA-approved cell therapy products (2010-2024) provides crucial insights into real-world potency test implementation [14]. This analysis identified 104 total potency tests used for these products, with 32% redacted due to proprietary information [14].

Table 2: Potency Test Measurements for 31 FDA-Approved Cell Therapy Products (2010-2024)

Category of Potency Test	Number of Tests	Percentage of Non-Redacted Tests	Examples of Specific Measurements
Viability and Count	37 tests	52%	Total nucleated cells (TNC), viability of CD45+ cells, viable CD34+ cell count [14]
Expression	19 tests	27%	CAR expression by flow cytometry, gene or protein expression [14]
Bioassays	7 tests	7%	Interferon-γ production upon stimulation with target cells, colony forming unit (CFU) assay [14]
Genetic Modification	6 tests	9%	Vector copy number (VCN), percent LVV+ cells [14]
Histology	2 tests	3%	Tissue organization, viability & retention of important cell types [14]

Key findings from this analysis include:

• Each CTP utilized an average of 3.4 potency tests (standard deviation 2.0) [14].
• "Viability and count" and "Expression" were most frequently used together, occurring for 16 CTPs (52%) [14].
• 26 of the 31 CTPs (84%) cited physicochemical assays (non-bioassays) as potency tests [14].

Comparative Potency Testing Expectations

Table 3: Direct Comparison of FDA and EMA Potency Testing Requirements

Aspect	FDA Expectations	EMA Expectations
Overall Approach	Comprehensive potency assurance strategy; validated functional potency assay essential for pivotal studies [8] [12]	Infectivity and transgene expression often sufficient in early phase; less functional assays may be acceptable later [12]
Number of Assays	Multiple potency assays may be requested; draft guidance suggests multifaceted approach [10] [8]	Generally accepts one robust potency assay addressing main mechanism of action [10]
Phase Appropriateness	Acknowledged in draft guidance, but industry seeks more clarity for early-phase development [10]	More flexible early-phase expectations with increasing rigor through development [9]
Viral Vectors for In Vitro Use	Classifies as drug substance; expects more functional assays [12]	Considers as starting materials; generally accepts infectivity and expression assays [12]

The American Society of Gene & Cell Therapy has advocated for a risk-based approach including "the development of one robust in vitro potency assay addressing the main mechanism of action of the final drug product for product release," with additional measures serving as characterization assays without strict acceptance criteria [10].

Experimental Protocols and Methodologies for Potency Testing

Potency Assay Development Lifecycle

The development of potency assays for CGT products typically follows a structured, phased approach that aligns with product development stages [4].

Diagram 1: Potency Assay Development Lifecycle

Key Experimental Protocols

CAR T-cell Potency Assay Protocol

The FDA-approved CAR T-cell products (Kymriah, Yescarta) utilize potency assays measuring interferon-γ production in response to target cells, demonstrating a functional bioassay approach [14].

Protocol:

• Stimulation: Co-culture CAR T-cells with CD19-expressing target cells (for anti-CD19 CAR T-cells) at optimized effector:target ratios [14].
• Incubation: Incubate for 16-24 hours under standard cell culture conditions (37°C, 5% CO₂).
• Cytokine Measurement: Harvest supernatant and quantify IFN-γ release using ELISA or multiplex immunoassay.
• Data Analysis: Calculate relative potency using parallel-line analysis or parallel-logistic analysis by comparing test samples to reference standard [4].

Technical Considerations:

• Cell Line Selection: Use target cells with consistent, physiologically relevant antigen expression levels.
• Controls: Include positive controls (reference CAR T-cell batch) and negative controls (non-transduced T-cells).
• Acceptance Criteria: Establish based on statistical analysis of historical data from multiple batches.

AAV Vector Potency Assay Protocol

For AAV-based gene therapies, potency assays typically measure transduction efficiency and transgene expression or function [4].

Protocol:

• Cell Seeding: Plate appropriate permissive cell line (considering AAV serotype tropism) at optimized density.
• Transduction: Incubate cells with serial dilutions of AAV vector reference standard and test samples.
• Gene Expression Analysis: After 48-72 hours, measure transgene expression:
  • Protein-based: Quantify protein expression via ELISA, western blot, or flow cytometry.
  • Functional: Measure enzymatic activity if transgene encodes an enzyme.
  • RNA-based: Quantify vector-derived RNA via RT-qPCR.
• Dose-Response Analysis: Generate dose-response curves and calculate relative potency using appropriate statistical models [4].

Statistical Analysis Methods for Potency Determination

Several statistical models are commonly used to analyze potency assay data [4]:

• Parallel-Logistic Analysis: Uses a 3-, 4-, or 5-parameter logistic regression model to generate a dose-response curve, allowing precise calculation of relative potency between test and reference samples.

• Parallel-Line Analysis: Employs linear regression to evaluate relative potency based on parallel dose-response relationships across different concentrations.

• Slope-Ratio Analysis: Also uses linear regression model but compares slopes of dose-response curves.

The choice of analysis method depends on the nature of the assay, the response curve characteristics, and the required degree of precision [4].

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 4: Key Research Reagents for CGT Potency Assay Development

Reagent/Category	Function in Potency Testing	Specific Examples & Considerations
Cell Lines	Provide cellular substrate for in vitro potency assays; critical for assessing biological response	- Permissive cell lines matching vector tropism- Engineered reporter cell lines- Primary cells when relevant [4]
Reference Standards	Serve as comparators for relative potency calculations; essential for assay calibration	- Well-characterized drug substance batch- International standards when available- Must be stable and representative [4]
Cytokine Detection Kits	Quantify functional responses in immune cell therapy potency assays	- IFN-γ ELISA for CAR T-cell potency- Multiplex immunoassays for broader cytokine profiling- Validated for sensitivity and dynamic range [14]
Flow Cytometry Reagents	Measure surface markers, CAR expression, and intracellular targets	- Fluorochrome-conjugated antibodies against CAR constructs- Viability dyes- Compensation controls and standardization beads [14]
Molecular Biology Kits	Quantify genetic modification and transgene expression	- qPCR/RTPCR for vector copy number and RNA expression- ddPCR for absolute quantification- Validated primer/probe sets [14]
Viral Vector Systems	Critical for gene therapy potency assay development	- AAV reference standards- Appropriate controls for infectivity and expression- Well-characterized viral stocks [4]

The regulatory landscape for CGT potency testing continues to evolve rapidly, with the FDA's draft guidance on Potency Assurance representing a significant shift toward a more comprehensive, risk-based approach [8]. For developers seeking global approval, key strategic considerations include:

• Engage Regulators Early: Pursue early regulatory interactions through FDA Type B meetings and EMA Scientific Advice to identify and address potential divergences in potency expectations [9].

• Implement Phase-Appropriate Strategies: Develop potency assays that evolve in complexity and rigor throughout the product lifecycle, recognizing that fully validated assays may not be feasible in early development phases [10].

• Design for Global Compliance: Create potency testing strategies that can adapt to both FDA and EMA expectations, particularly regarding the number and type of potency assays and the acceptance of mechanism-of-action-based functional assays [12].

• Leverage Real-World Data: Analyze potency testing approaches from approved products (Table 2) to inform assay selection and design, while recognizing that regulatory expectations continue to advance [14].

As global regulators push toward harmonization, maintaining current awareness of evolving guidance documents and adopting flexible, science-based potency assurance strategies will be essential for successfully navigating both FDA and EMA requirements and bringing transformative CGT products to patients worldwide.

The European Medicines Agency (EMA) regulates innovative medicinal products based on genes, cells, or tissues under the centralized framework for Advanced Therapy Medicinal Products (ATMPs) [15]. The legal basis for this framework is primarily defined by Regulation (EC) No 1394/2007, which established a dedicated committee, the Committee for Advanced Therapies (CAT), to provide specialized scientific assessment of these complex therapies [16]. The core objective of this regulatory structure is to ensure that pioneering treatments meet consistent standards of quality, safety, and efficacy across the European Union while fostering innovation in this rapidly evolving field [15] [16].

ATMPs are categorized into three main types, with an additional category for combination products, as detailed in Table 1 below [15].

Table 1: Classification of Advanced Therapy Medicinal Products (ATMPs) in the EU

ATMP Category	Core Definition	Key Examples
Gene Therapy Medicines	Contain genes that lead to a therapeutic, prophylactic, or diagnostic effect. They work by inserting 'recombinant' genes into the body [15].	Treatments for genetic disorders, cancer, or long-term diseases [15].
Somatic-Cell Therapy Medicines	Contain cells or tissues that have been manipulated to change their biological characteristics or are not intended for the same essential function in the body [15].	Cells used to cure, diagnose, or prevent diseases through repaired or altered biological functions [15].
Tissue-Engineered Medicines	Contain cells or tissues that have been modified to repair, regenerate, or replace human tissue [15].	Products for cartilage repair, skin regeneration, or other structural tissue restoration [15].
Combined ATMPs	Contain one or more medical devices as an integral part of the medicine [15].	Cells embedded in a biodegradable matrix or scaffold [15].

The regulatory pathway for ATMPs is predominantly centralized, requiring a single marketing authorization application evaluated by the EMA and approved by the European Commission [15]. The CAT is instrumental in this process, conducting the initial scientific assessment and preparing a draft opinion on quality, safety, and efficacy for the Committee for Medicinal Products for Human Use (CHMP) [15]. Furthermore, the EMA supports developers through various means, including scientific guidelines, an ATMP classification procedure, and specific support for micro-, small-, and medium-sized enterprises (SMEs) via a certification procedure for quality and non-clinical data [15] [16].

Key EMA Guidelines on Potency Testing for Cell-Based Immunotherapy

Potency is a Critical Quality Attribute (CQA) that represents the quantitative measure of the biological activity of a biological medicinal product and is expected to be linked to its clinical effect [17]. For cell-based immunotherapies, potency testing is particularly challenging due to the complex and living nature of the product, which often involves multiple mechanisms of action [17].

The overarching guideline for these products is the "Guideline on human cell-based medicinal products" (EMEA/CHMP/410869/2006) [18]. However, for immunotherapies targeting cancer, the most specific and critical guidance is the "Guideline on potency testing of cell based immunotherapy medicinal products for the treatment of cancer" (CHMP/BWP/271475/06) [19] [18]. This guideline establishes that assays measuring only viability and/or cell phenotype are insufficient for demonstrating potency; instead, the potency assay must be a quantitative biological assay that reflects the product's intended Mechanism of Action (MoA) [17].

Table 2: Summary of Key EMA Guidelines Relevant to Potency Testing of Cell-Based Immunotherapies

Guideline Title	Reference Code	Primary Focus and Relevance
Guideline on potency testing of cell based immunotherapy medicinal products for the treatment of cancer	CHMP/BWP/271475/06 [18]	Provides specific requirements for developing a potency assay that reflects the biological activity relevant to destroying cancer cells [19] [18].
Guideline on human cell-based medicinal products	EMEA/CHMP/410869/2006 [18]	The overarching quality guideline for cell-based medicines, outlining general principles for quality, safety, and efficacy, including potency [18].
Guideline on quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells	CAT/CHMP/GTWP/671639/2008 [18]	Specifically addresses products like CAR-T cells, where cells are genetically modified, impacting the strategy for potency testing [17] [18].
Reflection paper on stem cell-based medicinal products	EMA/CAT/571134/2009 [18]	Provides considerations for the development of stem cell-based products, which may be relevant for certain immunotherapies [18].
ICH Q6B: Specifications: Test procedures and acceptance criteria for biotechnological/biological products	CPMP/ICH/365/96 [18]	An international guideline adopted in the EU that defines general principles for setting specifications for biological products, including the role of potency [17].

The development of a potency assay must be mechanism-based. For a CAR-T cell product, for instance, the biological activity depends on a chain of events: cell viability, transduction efficiency (number of vector copies per cell), CAR transgene expression, and the final cytotoxic activity against target tumor cells [17]. A robust potency testing strategy should therefore ideally capture all these facets, potentially requiring a combination of assay formats.

The EMA acknowledges the technical challenges in developing a single, validated functional potency assay for such complex products. Therefore, while a quantitative functional assay is the goal, the regulatory approach can be pragmatic. The EMA guidances allow for the use of a validated surrogate assay for product release, provided that a functional assay is available for characterization and that a correlation between the two assays has been demonstrated [17]. This is a notable point of potential divergence from the U.S. Food and Drug Administration (FDA), which typically insists on a quantitative functional potency assay for lot release [17].

Comparative Analysis: EMA vs. FDA Regulatory Requirements

Navigating the regulatory landscape for cell-based immunotherapies requires a clear understanding of the similarities and differences between the two major regulatory bodies: the EMA and the FDA. While there is a strong drive for global regulatory convergence, significant differences in approach and specific requirements remain, which can impact global development strategies [20].

Table 3: Comparison of EMA and FDA Approaches to Cell-Based Immunotherapy Potency Testing

Aspect	European Medicines Agency (EMA)	U.S. Food and Drug Administration (FDA)
Overarching Principle	Potency is the quantitative measure of biological activity, linked to the relevant biological properties [17].	Potency is the specific ability or capacity of the product to effect a given result [17].
Key Guideline(s)	- Guideline on potency testing of cell based immunotherapy medicinal products for the treatment of cancer (CHMP/BWP/271475/06) [18]- Guideline on human cell-based medicinal products (EMEA/CHMP/410869/2006) [18]	- Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (2011) [11] [17]- Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products (2024) [11]
Core Assay Expectation	A quantitative biological assay reflecting the Mechanism of Action (MoA) is required. A validated surrogate assay may be acceptable for release if correlated with a functional characterization assay [17].	A quantitative functional potency assay that measures the biological activity is expected for lot release [17].
GMP Compliance	Mandatory GMP compliance for all clinical trial stages, verified through self-inspections and member state oversight [20].	Phase-appropriate cGMP compliance, with full compliance verified via pre-license inspection (BLA stage); relies on attestation in early phases [20].
Donor Eligibility	General guidance referencing compliance with EU and member state legal requirements; limited specific direction on emerging pathogens [20].	Highly prescriptive requirements for donor screening, testing, laboratory qualifications, and restrictions on donor pooling [20].

A central point of comparison lies in the potency assay requirement. As noted in Table 3, the FDA maintains a firm stance on the need for a quantitative functional assay for product release [17]. In contrast, the EMA provides more flexibility, allowing for a surrogate method for release testing provided it is justified and correlated with a biological function [17]. This difference has had practical consequences; for example, the development of the tumor-infiltrating lymphocyte (TIL) therapy Lifileucel faced challenges in meeting the FDA's requirements for a functional potency assay [17].

Another key difference is in the approach to GMP compliance. The EU system mandates adherence to GMP standards from the earliest clinical trials, with compliance ensured through a system that includes mandatory self-inspections [20]. The FDA, however, employs a risk-based, phase-appropriate approach, where full cGMP compliance is expected primarily at the time of commercial licensure and is verified via a pre-approval inspection [20].

Furthermore, requirements for determining allogeneic donor eligibility are more detailed and prescriptive from the FDA, which specifies the exact communicable disease agents to test for, the testing methods, and the qualifications of the testing laboratories [20]. The EMA guidelines, while acknowledging the importance of this issue, direct developers to comply with the relevant EU and national laws without providing the same level of specific, centralized direction [20].

The Scientist's Toolkit: Essential Reagents and Materials for Potency Assays

Developing a robust potency assay for cell-based immunotherapies requires a suite of specialized reagents and analytical tools. The selection is guided by the product's MoA and the specific assay format chosen. Below is a non-exhaustive list of key solutions and their functions.

Table 4: Key Research Reagent Solutions for Potency Assay Development

Reagent / Material	Critical Function in Potency Testing
Target Cells	Engineered cell lines or primary cells expressing the target antigen; used as the biological target in co-culture functional assays to measure product activity (e.g., cytotoxicity) [17].
Cytokine Detection Kits	ELISA, MSD, or Luminex-based kits to quantify cytokine secretion (e.g., IFN-γ, IL-2) upon target cell engagement, serving as a measure of T-cell activation [17].
Flow Cytometry Reagents	Fluorochrome-conjugated antibodies for detecting cell surface markers (e.g., CAR expression, activation markers) and intracellular proteins; vital for phenotyping and transduction efficiency [17].
Cell Viability & Cytotoxicity Assays	Reagents for assays like Real-Time Cell Analysis (RTCA) or measurement of lactate dehydrogenase (LDH) release to quantitatively measure target cell killing [17].
Reference Standards	A well-characterized cell batch used as a comparator in relative potency assays to ensure consistency between batches and over time [17].
Cell Culture Media & Supplements	Serum-free media, cytokines (e.g., IL-2, IL-7, IL-15), and other supplements essential for maintaining cell viability and function during the assay period [17].

Experimental Protocols for Potency Assay Development

This section outlines a generalized experimental workflow for developing and validating a cell-based potency assay, such as a cytotoxicity assay for a CAR-T product. The protocol is based on regulatory expectations for reflecting the product's MoA [17].

Protocol: Cytotoxicity Assay for CAR-T Cell Potency

Principle: This assay measures the specific lytic activity of CAR-T cells against antigen-positive target cells. The percentage of target cell killing is quantified over a defined co-culture period, providing a direct measure of the product's effector function.

Key Materials:

• Effector cells: The CAR-T cell product under test.
• Target cells: Two populations: antigen-positive cells and antigen-negative cells (as a control for specificity).
• Cell culture plates: 96-well plates suitable for the detection method.
• Detection system: Real-time cell analyzer (RTCA) or LDH release detection kit.
• Culture medium: Appropriate assay medium without phenol red if colorimetric detection is used.

Methodology:

• Preparation: Thaw and rest effector and target cells as per standardized protocols. Count and resuspend cells in assay medium.
• Plate Setup: Seed target cells in the plate. For RTCA, background measurements are taken before adding effector cells.
• Effector-Target Co-culture: Add effector cells to the target cells at multiple Effector to Target (E:T) ratios (e.g., 20:1, 10:1, 5:1, 1:1). Include control wells for:
  • Target cells alone (spontaneous control).
  • Target cells with lysis buffer (maximum release control).
  • Effector cells alone (effector background control).
  • Co-culture of effector cells with antigen-negative target cells (specificity control).
• Incubation: Incubate the plate under standard culture conditions (37°C, 5% CO2) for the predetermined assay duration (e.g., 24-48 hours).
• Measurement: Quantify cytotoxicity according to the detection system:
  • For RTCA: Measure cell impedance continuously or at endpoint. Calculate percent cytotoxicity as (1 - (Impedance of co-culture well / Impedance of target cell control well)) * 100.
  • For LDH Release: Measure LDH activity in the supernatant. Calculate percent cytotoxicity using the formula: ((Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)) * 100.
• Data Analysis: Plot dose-response curves (percent cytotoxicity vs. E:T ratio). The relative potency of a test sample can be calculated by comparing its dose-response curve to that of a reference standard using parallel-line analysis or similar statistical methods [17].

Validation Parameters: Once the method is developed, it must be validated for its intended purpose. Key validation parameters include accuracy, precision (repeatability and intermediate precision), linearity, range, specificity, and robustness according to ICH Q2(R1) principles [17] [18].

The EMA's regulatory framework for ATMPs provides a structured yet adaptable pathway for the development of cell-based immunotherapies. The cornerstone of quality assessment within this framework is a mechanism-based, quantitative potency assay, as detailed in specialized guidelines like the one for cancer immunotherapies. While the EMA and FDA share the fundamental goal of ensuring product efficacy and patient safety, key strategic differences exist. Developers must be aware of these distinctions, particularly regarding the flexibility in potency assay strategies, GMP implementation timelines, and donor eligibility requirements. A deep understanding of both regulatory landscapes is not merely a compliance exercise but a critical enabler for designing efficient global development strategies that can bring transformative cell-based immunotherapies to patients in a timely and safe manner.

Comparative Analysis of Core Regulatory Philosophies and Definitions

The development and approval of cell therapy products (CTPs) are governed by distinct regulatory philosophies in the United States (US) and European Union (EU). The US Food and Drug Administration (FDA) regulates these products as biologics, requiring a potency test for release testing as part of licensure to ensure the product can achieve its intended mechanism of action, assess manufacturing consistency, and evaluate product stability [3]. The European Medicines Agency (EMA) classifies these therapies as Advanced Therapy Medicinal Products (ATMPs), a category encompassing gene therapy medicinal products, somatic cell therapy medicinal products, tissue-engineered products, and combined ATMPs [20]. While both agencies share the fundamental goal of ensuring patient safety and product efficacy, their approaches to demonstrating potency—a quantitative measure of a product's biological activity—diverge in significant ways, creating a complex landscape for global drug development [9].

A recent analysis of clinical trial data submitted to both agencies revealed that only 20% of the information matched, highlighting major inconsistencies in regulatory expectations [9]. This divergence can lead to approval delays, increased costs, and complex hurdles for developers aiming for global market access. This guide provides a detailed, objective comparison of the core regulatory philosophies and definitions governing cell therapy potency testing, equipping researchers and drug development professionals with the data needed to navigate these critical requirements.

Comparative Analysis of Regulatory Philosophies and Requirements

The regulatory pathways for cell therapies in the US and EU differ in their foundational structure, terminology, and specific technical requirements. Understanding these differences is crucial for strategic planning.

Foundational Frameworks and Definitions

The FDA's Center for Biologics Evaluation and Research (CBER) provides a structured framework for CTPs. A key differentiator is the FDA's classification of products as either in vivo gene therapy (where genetic material is delivered directly to a patient's cells) or ex vivo gene therapy/cell therapy (where cells are modified outside the body before transplantation) [21]. In contrast, the EMA operates under the ATMP framework defined by Regulation (EC) No 1394/2007, which uses different categorical definitions for gene therapies and cell therapies [9] [21]. This fundamental difference in nomenclature and categorization is the first major point of divergence sponsors must recognize.

Key Regulatory Differences for Cell Therapy Products

Table 1: Core Regulatory Differences Between FDA and EMA for Cell Therapies

Aspect	US FDA Approach	EMA Approach
Regulatory Classification	Biologics; differentiated as in vivo or ex vivo gene therapy/cell therapy [21]	Advanced Therapy Medicinal Products (ATMPs) [9]
Marketing Authorization	Biologics License Application (BLA) demonstrating safety, purity, and potency [9]	Marketing Authorization Application (MAA) under ATMP framework [9]
Potency Testing Philosophy	Requires a potency test for product release; accepts a potency assay matrix [3] [22]	Expects a risk-based approach; guidance consolidated in multidisciplinary ATMP guideline [20]
Starting Materials	Does not typically require plasmids to be developed under full GMP [21]	Requires plasmids used to generate vectors to be developed under Good Manufacturing Practice (GMP) [21]
Donor Eligibility & Testing	Highly prescriptive requirements for donor screening and testing for communicable diseases [20]	General guidance provided; must comply with relevant EU and member state-specific legal requirements [20]
GMP Compliance	Phase-appropriate attestation, with full compliance verified via pre-license inspection [20]	Mandatory self-inspections and compliance with GMP specific to ATMPs are prerequisites for clinical trials [20]
Environmental Risk	Not a primary focus in reviewed documents	Requires an Environmental Risk Assessment (ERA) for products containing or consisting of GMOs [21]

Quantitative Analysis of FDA-Approved Product Potency Tests

An analysis of the 31 US FDA-approved CTPs from 2010-2024 provides tangible data on what the agency has accepted for potency testing. This dataset offers valuable, real-world insights for developers designing their potency strategy.

Table 2: Analysis of Potency Tests for 31 FDA-Approved Cell Therapy Products (2010-2024) [3]

Category of Potency Test	Number of Tests (Non-Redacted)	Percentage of Total Non-Redacted Tests	CTPs Using This Test Type
Viability and Count	37	52%	19 of 31 (61%)
Expression	19	27%	20 of 31 (65%)
Bioassays	7	7%	7 of 31 (23%)
Genetic Modification	6	9%	Not Specified
Histology	2	3%	Not Specified
Total (Non-Redacted)	71	100%

This analysis reveals several key patterns [3]:

• A total of 104 potency tests were reported for the 31 CTPs, with 33 (32%) redacted for proprietary reasons.
• The average number of potency tests per CTP is 3.4 (standard deviation 2.0), with a median of 3.0. The highest number of tests for a single product was 8.
• The number of potency tests per product has not shown a significant increasing trend over time, indicating consistency in regulatory expectations regarding the number of tests.
• The two most frequently used test categories, "Viability and Count" and "Expression," were used together for 16 CTPs (52%).
• While only 23% of CTPs publicly reported a bioassay, due to redactions, as many as 77% could potentially have one, and 84% cited physicochemical assays as a potency test.

Experimental Protocols for Potency Assay Development

Developing robust potency assays is a central requirement of both regulatory bodies. The following experimental workflows and methodologies are critical for generating acceptable data.

Potency Assay Development Lifecycle and Methodology

The development of a potency assay follows a defined lifecycle of development, qualification, and validation [4]. The methodology must be tailored to the product's specific Mechanism of Action (MOA).

Experimental Workflow for Cell-Based Potency Assays:

• Cell Line Selection: Choose a cell line based on vector tropism (for gene therapies) and compatibility with the vector's promoters and the therapy's MOA. Proper cell banking and testing are essential for reproducibility [4].
• Transduction/Culture Conditions: Optimize conditions for introducing the therapy to the cells (e.g., transduction multiplicity of infection) or for culturing the therapeutic cells themselves.
• Experimental Readout: Measure a relevant downstream biological response. This can include:
  • Nucleic Acid-Based: Quantification of vector-derived RNA using RT-qPCR [14].
  • Protein-Based: Measurement of protein expression (e.g., CAR expression by flow cytometry) [3] [14] or enzymatic activity.
  • Functional/Cell-Based: Measurement of cytokine release (e.g., IFNγ in response to target cells) [3] [14] or cytotoxic activity (e.g., via cytotoxicity assay with flow cytometry) [14].
• Analysis Method: Employ statistical models to determine relative potency. Common methods include parallel-logistic analysis (using a 3-5 parameter logistic regression model), parallel-line analysis, and slope-ratio analysis [4].

Figure 1: Potency Assay Development Workflow and Lifecycle

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Potency Assay Development

Reagent/Material	Function in Potency Testing
Cell Lines	Provides a biologically relevant system for measuring the product's functional activity. Must be selected for compatibility with the product's mechanism of action [4].
Flow Cytometry Antibodies	Enables quantification of specific cell surface (e.g., CAR expression) and intracellular markers for identity, purity, and potency assessments [3] [14].
qPCR/RT-qPCR Reagents	Used for quantifying vector copy number (VCN) and transgene expression levels, serving as a direct measure of genetic modification potency [14].
ELISA/Cytokine Detection Kits	Measures the secretion of specific proteins (e.g., IFNγ) in response to stimulus, providing a quantitative readout of cell product activation and function [3] [14].
Cell Viability Assays	Distinguishes live from dead cells (e.g., using dye exclusion), a fundamental and commonly used potency parameter for cell-based products [3].
Cell Culture Media & Supplements	Supports the growth and maintenance of the therapeutic cells or the assay cell line, ensuring consistent and reproducible assay performance [4].

Analysis of Divergent Regulatory Expectations

The practical implications of regulatory divergence are significant and impact nearly every stage of product development.

Strategic Regulatory Engagement Pathways

Engaging with regulators early is critical, but the preferred mechanisms differ. The FDA offers Type B meetings (e.g., pre-IND, EOP) for structured feedback [9]. The EMA provides a Scientific Advice procedure, which can be sought at the national level from a specific Member State or centrally from the EMA's Committee for Medicinal Products for Human Use (CHMP) [9] [20]. A proactive strategy involves engaging both agencies in parallel to understand their specific expectations and identify potential conflicts early in development.

Figure 2: Strategic Regulatory Engagement Pathways

Navigating Potency-Specific Challenges

A primary challenge in potency testing is the lack of global harmonization, which forces sponsors to prepare distinct applications and adapt trial protocols for each agency [9]. The American Society of Gene and Cell Therapy (ASGCT) has highlighted that challenges associated with potency assays frequently lead to development delays, including clinical holds [22]. To mitigate this, developers should:

• Adopt a Phase-Appropriate Strategy: Early in development, focus on gaining product and process understanding. A single, robust in vitro potency assay addressing the main MOA may be sufficient for release, with additional assays used for characterization [22]. Full validation is expected later to support marketing applications [4].
• Implement a Risk-Based Approach: Align the potency assurance strategy with existing quality risk management systems (e.g., ICH Q9) to avoid redundant documentation [22].
• Plan for Comparability: Anticipate manufacturing changes and keep samples from early batches to conduct comparability assessments, which are expected by both CBER and EMA [21].

The regulatory philosophies of the FDA and EMA regarding cell therapy potency testing, while aligned in their ultimate goals of safety and efficacy, present a landscape of defined divergence. The FDA's approach is often characterized by structured pathways and phase-appropriate flexibility, whereas the EMA demands more extensive and upfront GMP compliance and data, often with a focus on centralized guideline adherence. Quantitative analysis of approved products reveals that developers have successfully navigated these requirements by employing a multi-faceted testing strategy, heavily relying on viability, count, and expression assays.

For researchers and drug development professionals, success in this environment requires a proactive, nuanced strategy. This involves early and parallel engagement with both agencies, a deep understanding of the specific and sometimes divergent technical requirements (from donor eligibility to starting materials), and the implementation of a robust, phase-appropriate, and risk-based potency assurance plan. By recognizing these differences as integral to global development planning rather than as obstacles, sponsors can streamline approvals and accelerate the delivery of transformative cell therapies to patients worldwide.

The Importance of Early Regulatory Intelligence in Global Planning

For developers of cell and gene therapy products (CGTs), navigating the divergent regulatory expectations for potency testing between the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) presents a significant challenge. Potency testing, which measures the biological activity of a therapy and its specific ability to achieve its intended effect, represents a universal requirement for market approval [5] [23]. However, the strategic importance of understanding the nuanced differences between major regulatory agencies from the earliest stages of development cannot be overstated. Early regulatory intelligence—the systematic gathering and analysis of regulatory requirements—enables developers to design a unified global strategy that balances speed to market with comprehensive compliance, ultimately accelerating patient access to transformative therapies.

The stakes for misalignment are high. Insufficient potency assurance can lead to clinical holds, development delays, and failure to secure market authorization in key regions [5] [10]. This is particularly critical for therapies targeting ultra-rare diseases, where sufficient trial populations may not be available within a single regulatory jurisdiction, making global planning a necessity rather than an option [5]. This guide provides a comparative analysis of EMA and FDA requirements for cell therapy potency testing, supported by experimental data and strategic frameworks to inform early-stage planning.

Comparative Analysis of EMA and FDA Regulatory Frameworks

Organizational Structures and Philosophical Approaches

The foundational differences between the EMA and FDA stem from their distinct organizational structures and regulatory philosophies, which directly influence their approach to potency assurance.

• FDA: Centralized Federal Authority - The FDA operates as a centralized federal agency with direct decision-making power. Its structure enables relatively swift decision-making, with review teams composed of full-time FDA employees. Once the FDA approves a therapy, it is immediately authorized for marketing throughout the entire United States [24].

• EMA: Coordinated European Network - In contrast, the EMA functions as a coordinating body rather than a direct decision-making authority. It oversees a network of national competent authorities across EU Member States. The Committee for Medicinal Products for Human Use (CHMP) conducts scientific evaluations, but the final legal authority to grant marketing authorization rests with the European Commission. This network model incorporates broader scientific perspectives but requires more complex coordination across diverse healthcare systems [24].

Key Regulatory Guidance Documents

Both agencies have established comprehensive guidance documents specifically addressing potency testing for advanced therapies, though with differing emphases and requirements.

Table 1: Key Potency Testing Guidelines from FDA and EMA

Agency	Guidance Document	Key Focus Areas	Notable Requirements
FDA	Potency Assurance for Cellular and Gene Therapy Products (Draft, 2023) [11]	Multi-faceted potency assurance strategy, phase-appropriate development, risk management	Recommends a comprehensive strategy beyond a single potency assay; emphasizes product and process understanding
EMA	Guideline on potency testing of cell-based immunotherapy medicinal products for the treatment of cancer (2006, revised) [19] [18]	Cell-based immunotherapy for cancer; implementation of 3Rs (Replacement, Reduction, Refinement)	Specific focus on immunotherapies; emphasizes appropriate methods for different product types
EMA	Guideline on human cell-based medicinal products (2006) [18]	Overarching requirements for all cell-based therapies	Defines general quality, non-clinical, and clinical standards for cell-based products

Direct Comparison of Potency Testing Requirements

A detailed analysis reveals critical divergences in how the two agencies approach potency testing, particularly regarding timing, validation, and strategic implementation.

Table 2: Direct Comparison of EMA and FDA Potency Testing Requirements

Aspect	U.S. FDA	European EMA
Method Validation Timing	Validation typically not required for Phase 1 INDs [5]	"An appropriate degree of method validation should be applied at each stage"; safety-relevant assays must be fully validated [5]
Strategic Approach	Multi-faceted "potency assurance strategy" encouraged [10]	Flexible approach, but relies on well-established guidelines for specific product categories [18]
Number of Assays	Draft guidance suggests multiple assays, though industry advocates for one robust assay for release [10]	Expectations vary by product type; specific guidelines exist for immunotherapies and gene therapies [18]
Critical Divergence	More flexible early-phase validation	Explicit validation expectations even for early-phase trials
Industry Concern	Potential for significant development burden with limited benefit [10]	Smaller national agencies may apply guidelines verbatim, limiting flexibility [5]

The diagram below illustrates the divergent regulatory pathways and key decision points for potency strategy development facing global developers.

Analytical Methods and Experimental Approaches for Global Compliance

Current Landscape of Potency Assays from Approved Products

Analysis of FDA-approved cell therapy products (CTPs) reveals the current state of potency testing in regulatory practice. A 2025 review of the 31 U.S. FDA-approved CTPs identified 104 total potency tests across these products, averaging 3.4 potency tests per product [25]. The distribution of these tests demonstrates the diverse methodological approaches required for comprehensive product characterization.

Table 3: Distribution of Potency Test Types for 31 FDA-Approved Cell Therapy Products

Potency Test Category	Number of Tests	Percentage of Total	Example Methodologies
Viability and Count	37	52%	Cell counting, viability staining (e.g., trypan blue exclusion)
Expression	19	27%	Flow cytometry for surface markers, qPCR for gene expression
Bioassays	7	7%	Cytokine secretion (e.g., IFN-γ ELISpot), cytotoxicity assays
Genetic Modification	6	9%	Vector copy number (VCN) by ddPCR, integration site analysis
Histology	2	3%	Tissue structure assessment, staining evaluation

Notably, 61% of CTPs used cell viability or count as a potency test, while 65% employed gene or protein expression measurements [25]. The most common combination was "Viability and Count" with "Expression" tests, occurring for 16 CTPs (52%). However, a significant portion of potency tests (32%) were redacted from public documents, suggesting that potentially sensitive or proprietary bioassays may be more commonly employed than publicly available data indicates [25].

Advanced Methodologies for Next-Generation Potency Testing

As cell therapies grow more complex, particularly with the rise of CAR-T products, potency assays have evolved beyond simple viability measures to incorporate multi-omics approaches that better reflect mechanism of action (MoA).

CAR-T Cell Potency Assessment: For CAR-T products, potency is typically assessed through a matrix of assays evaluating three key functional domains [26]:

• Immediate Effector Function: Cytotoxicity, cytokine release (IFN-γ, TNF-α, IL-2), and degranulation markers (CD107a/LAMP1)
• Viability and Expansion Capacity: Cell proliferation and viability measurements
• Persistence Potential: Phenotype characterization (naïve, memory, exhausted subsets), in vivo tracking, and CAR transgene expression

Multi-Omics Profiling for Enhanced Characterization: Advanced analytical approaches provide deeper product understanding, though they are typically used for characterization rather than lot release [26]:

• Genomic Profiles: Vector copy number (VCN) quantification by droplet digital PCR (ddPCR), vector integration site analysis using pipelines like INSPIIRED or EpiVIA, and T-cell receptor (TCR) repertoire sequencing to assess clonal diversity [26].

• Epigenomic Profiles: DNA methylation analysis and chromatin accessibility mapping (ATAC-seq) to characterize differentiation states and persistence potential [26].

• Transcriptomic and Proteomic Profiles: Bulk and single-cell RNA sequencing to identify transcriptional subtypes, and protein expression analysis to quantify critical effector molecules [26].

The following workflow illustrates how these advanced methodologies integrate into a comprehensive potency assessment strategy for sophisticated cell therapies like CAR-T products.

The Scientist's Toolkit: Essential Reagents and Technologies

Implementing robust potency assays requires specific research reagents and technological platforms. The table below details essential solutions for developing and executing cell therapy potency tests.

Table 4: Essential Research Reagent Solutions for Potency Assay Development

Reagent/Technology	Function in Potency Testing	Application Examples
ddPCR/qPCR Systems	Absolute quantification of vector copy number (VCN) and transgene expression	Quality control release testing for genetically modified CTPs [26] [25]
Flow Cytometry Panels	Multiplexed measurement of cell surface markers, intracellular cytokines, and phosphorylation states	Phenotypic characterization, detection of activation markers (e.g., CD69), and quantification of CAR expression [26] [25]
ELISA/ELISpot Kits	Quantification of secreted cytokines and other soluble factors	IFN-γ release assays for CAR-T potency; measurement of other cytokines (IL-2, TNF-α) [26] [23]
Single-Cell RNA Sequencing Kits	High-resolution transcriptomic profiling at single-cell level	Identification of critical cell subpopulations; correlation of transcriptional signatures with product potency [26]
Cytotoxicity Assay Reagents	Measurement of target cell killing capacity	Real-time cell analysis (e.g., xCelligence) or flow-based (e.g., Annexin V) cytotoxicity assays [26]
Cell Viability Stains	Discrimination of live/dead cells and functional status	Trypan blue exclusion for counting; multiplexed viability dyes for flow cytometry [25]

Strategic Implementation for Global Development

Developing a Phase-Appropriate Global Potency Strategy

The most critical consideration for global development is implementing a phase-appropriate strategy that satisfies both agencies while maintaining development efficiency. Key strategic principles include:

• Early Engagement and Roadmapping: Initiate regulatory dialogue early, particularly through FDA pre-IND meetings and EMA Scientific Advice procedures. Develop a "potency road map" that visualizes the progression of potency assays from early to late development phases [5] [24].

• Mechanism of Action (MoA)-Driven Design: Base potency assay selection on the proposed MoA, even when complete understanding may evolve during development. For CAR-T products, this means assays should reflect the key biological functions of target cell recognition, activation, and destruction [26] [23].

• Leverage Prior Knowledge: Utilize existing information from similar products or platform technologies. As noted by the American Society of Gene & Cell Therapy (ASGCT), "generalized information about acceptable potency assays could be shared by the Agency" for product classes like CAR-Ts [10].

• Integrated Quality Risk Management: Implement potency assurance within existing quality systems rather than creating separate documentation structures. Align with ICH Q9 (Quality Risk Management) and ICH Q10 (Pharmaceutical Quality System) to avoid redundant efforts [10].

Navigating Regulatory Divergence: Case Studies and Practical Considerations

Case Study: The TCID50 Assay Challenge The 50% tissue culture infectious dose (TCID50) assay, used in early-phase gene therapy programs to assess infectivity and potency, illustrates regulatory divergence. This classic virology method is "highly variable, not tissue-specific, and performed in a nonrepresentative system" [5]. While it is "required by some, but not all national regulatory agencies" as an early-phase potency measure, developers often implement it with the awareness that they will need to transition to more representative assays later in development—effectively "kicking the proverbial potency can down the method life cycle road" [5].

The Correlation Challenge: Potency vs. Efficacy A fundamental challenge in potency testing is the uncertain relationship between potency measurements and clinical outcomes. For Kymriah (tisagenlecleucel), the first FDA-approved CAR-T therapy, potency was defined as the ability to secrete interferon-γ (IFN-γ) upon encountering CD19+ target cells. However, FDA documentation noted that "IFN-γ production varied greatly from lot-to-lot, making it difficult to correlate IFN-γ production in vitro to tisagenlecleucel safety or efficacy" [23]. This case highlights that while correlation between potency tests and clinical efficacy is desirable, it is not always achievable or required for approval, provided the product demonstrates an acceptable benefit-risk profile.

Early regulatory intelligence in cell therapy development is not merely an administrative exercise but a strategic imperative. The divergent pathways of the FDA and EMA regarding potency testing require careful navigation from the earliest stages of development. A successful global strategy incorporates:

• Proactive Planning: Anticipating regulatory differences rather than reacting to them
• Phase-Appropriate Implementation: Balancing flexibility for early-phase development with rigor for later phases
• Science-Driven Assay Design: Focusing on biological relevance to mechanism of action
• Unified Documentation: Integrating potency assurance within existing quality systems

By adopting this strategic approach, developers can create a cohesive potency assurance framework that satisfies both FDA and EMA requirements, minimizes development delays, and ultimately accelerates the delivery of transformative therapies to patients worldwide.

Developing a Compliant Potency Assay Strategy: From MoA to Testing Matrix

Linking Potency Assay Design to Mechanism of Action (MoA)

For cell therapy developers, designing potency assays that accurately reflect the product's Mechanism of Action is a critical regulatory requirement. Potency, defined as the "specific ability or capacity of the product to affect a given result," serves as the essential link between a therapy's quality attributes and its intended clinical effect. Regulatory agencies including the US Food and Drug Administration and the European Medicines Agency consider potency a Critical Quality Attribute that must be measured for each product lot to ensure consistent biological activity. The fundamental challenge lies in developing quantitative biological tests that capture the complex, multifaceted nature of cell therapies while satisfying distinct regulatory expectations across jurisdictions. This guide examines current practices, regulatory requirements, and experimental approaches for aligning potency assay design with therapeutic MoA to accelerate cell therapy development.

Quantitative Analysis of Approved Cell Therapy Potency Tests

A comprehensive analysis of potency tests used for the 31 US FDA-approved cell therapy products reveals clear patterns in current industry practices and regulatory acceptance. This data provides valuable benchmarking information for developers designing new potency assays.

Table 1: Distribution of Potency Test Types for 31 FDA-Approved CTPs [3]

Potency Test Category	Number of Tests	Percentage of Non-Redacted Tests	CTPs Using This Test Type
Viability and Count	37	52%	19 (61%)
Expression	19	27%	20 (65%)
Bioassays	7	7%	7 (23%)
Genetic Modification	6	9%	Not specified
Histology	2	3%	Not specified

Table 2: Statistical Analysis of Potency Testing for Approved CTPs [3]

Analytical Parameter	Value	Additional Context
Total Potency Tests	104	Across 31 CTPs
Redacted Tests	33 (32%)	Proprietary information
Non-Redacted Tests	71 (68%)	Available for analysis
Average Tests per CTP	3.4	Standard deviation 2.0
Median Tests per CTP	3.0	Q1: 1.5, Q3: 4.0
Maximum Tests for Single CTP	8	Lenmeldy
CTPs with Only One Test	8	26% of total

The data demonstrates that viability/count measurements and expression analyses constitute the majority (79%) of non-redacted potency tests used for approved cell therapy products. Notably, the average number of potency tests per product has remained stable over time, with no significant upward trend observed. This suggests that regulatory acceptance focuses on scientific justification rather than quantity of tests. Hematopoietic stem cell-cord blood products utilized the highest number of potency tests (average 4.4), while CAR-T products and tissue-engineered therapies employed fewer tests (averages of 1.9 and 1.8 respectively) [3].

Comparative Regulatory Frameworks: EMA vs. FDA

Key Regulatory Differences in Potency Testing Requirements

While both agencies share the fundamental goal of ensuring cell therapy safety and efficacy, their regulatory approaches exhibit important distinctions that impact potency assay strategy.

Table 3: EMA vs. FDA Regulatory Approaches to Cell Therapy Potency Testing [19] [11] [9]

Regulatory Aspect	FDA (US)	EMA (EU)
Overarching Guidance	Potency Assurance for Cellular and Gene Therapy Products (Draft, 2023)	Guideline on potency testing of cell-based immunotherapy medicinal products for the treatment of cancer (2006, revised)
Potency Assay Philosophy	Mechanism-of-action-based assays expected; "potency assay matrix" accepted for complex products	Strong emphasis on biologically relevant assays reflecting intended function; may require multiple parameters
Technical Standards	21 CFR Part 610.10 for potency testing of biologics	European Pharmacopoeia monographs relevant to ATMPs
Clinical Trial Requirements	IND application with proposed potency methods; 30-day review	CTA submission to National Competent Authorities; centralized via CTIS
Approval Pathways	BLA requiring demonstration of safety, purity, and potency	MAA under ATMP framework (Regulation EC No 1394/2007)
Expedited Programs	RMAT designation available	PRIME scheme for breakthrough ATMPs
Post-Marketing Surveillance	15+ years LTFU for gene therapies; REMS for high-risk products	Risk-based LTFU; mandatory RMPs for all ATMPs

Strategic Implications for Potency Assay Design

The divergence in regulatory approaches necessitates careful strategic planning for global development programs. The FDA has demonstrated greater flexibility in accepting real-world evidence and surrogate endpoints, particularly through expedited pathways like RMAT designation. In contrast, the EMA typically requires more comprehensive clinical data, emphasizing larger patient populations and long-term efficacy before granting approval [9]. This regulatory divergence means that a uniform approach to potency assay strategy is often insufficient, necessitating tailored applications to meet each agency's distinct expectations [9].

Industry feedback indicates that the FDA's draft guidance on potency assurance would benefit from clearer phase-appropriate requirements, particularly acknowledging that robust potency assays may not be feasible early in clinical development due to assay complexity, small sample sizes, and evolving mechanistic understanding [22]. Both agencies emphasize that potency testing should evolve throughout the product lifecycle, with increasing rigor and validation as development progresses toward marketing application.

MoA-Based Potency Assay Design: Methodologies and Protocols

Experimental Framework for MoA-Aligned Potency Assays

Designing potency assays that accurately reflect a cell therapy's Mechanism of Action requires a systematic approach to experimental design. The following workflow outlines the key methodological considerations for developing robust, MoA-based potency assays.

CAR-T Cell Therapy Potency Assay: A Case Study Protocol

Therapeutic MoA: Chimeric Antigen Receptor T-cells mediate target cell killing through antigen-specific recognition followed by activation of cytotoxic mechanisms.

Experimental Objective: Quantify CAR-T cell functional potency through measurement of target cell killing and associated activation markers.

Materials and Reagents:

• CAR-T cells: Final drug product or representative samples
• Target cells: Antigen-positive tumor cell lines (e.g., NALM-6 for CD19+ targets) or custom cell mimics (e.g., TruCytes)
• Culture media: Appropriate supplemented media for both cell types
• Detection reagents: Flow cytometry antibodies (CD107a, IFN-γ, Granzyme B), cell viability dyes, cytokine ELISA kits

Methodology:

• Co-culture Establishment:
  • Seed target cells in 96-well plates at optimized density (typically 1×10⁴ cells/well)
  • Add CAR-T cells at multiple effector-to-target ratios (e.g., 1:1, 5:1, 10:1)
  • Include appropriate controls (target cells alone, CAR-T cells alone, untransduced T-cells with targets)
  • Incubate at 37°C, 5% CO₂ for 18-24 hours

• Cytotoxic Activity Assessment:

  • Measure target cell viability using flow cytometry with viability dyes (e.g., propidium iodide, 7-AAD) or luminescent assays (e.g., CellTiter-Glo)
  • Calculate specific lysis percentage: % Specific Lysis = [1 - (Experimental Viability/Control Viability)] × 100

• Activation Marker Analysis:

  • Collect supernatant for cytokine analysis (IFN-γ, IL-2) via ELISA or multiplex immunoassay
  • Stain cells with fluorochrome-conjugated antibodies against CD107a, CD69, and intracellular cytokines for flow cytometric analysis
  • Fix and permeabilize cells for intracellular staining of effector molecules (Granzyme B, Perforin)

• Data Analysis and Potency Calculation:

  • Generate dose-response curves using CAR-T cell dilution series
  • Calculate relative potency compared to reference standard using parallel-line analysis or 4-parameter logistic models
  • Establish acceptance criteria based on validation data and reference standard performance

Validation Parameters: Assess assay precision (repeatability, intermediate precision), accuracy, linearity, range, robustness, and specificity according to ICH Q2(R1) guidelines [4] [27].

Essential Research Reagents and Tools for Potency Assay Development

The successful implementation of MoA-based potency assays requires carefully selected reagents and materials that ensure reproducibility and regulatory compliance.

Table 4: Essential Research Reagent Solutions for Cell Therapy Potency Assays [4] [27]

Reagent Category	Specific Examples	Function in Potency Assay	Key Selection Considerations
Reference Standards	In-house primary reference, WHO standards, commercially qualified standards	Calibrate potency measurements across batches and time	Well-characterized, sufficient quantity for long-term use, stability profile
Cell-Based Assay Systems	Tumor cell lines, primary cells, custom cell mimics (TruCytes)	Provide biological context for functional potency assessment	Antigen expression relevance, growth characteristics, genetic stability, documentation
Detection Reagents	Fluorochrome-conjugated antibodies, ELISA kits, luminescent substrates	Enable quantification of analytical endpoints	Specificity, sensitivity, lot-to-lot consistency, regulatory compliance (e.g., animal component-free)
Assay Platforms	Flow cytometers, plate readers, automated cell counters, HPLC/UPLC systems	Instrumentation for endpoint measurement	Validation status, software controls, calibration protocols, GMP compatibility
Culture Components	Serum-free media, cytokines, activation reagents, differentiation kits	Maintain relevant cellular functions during assay	Composition consistency, endotoxin levels, performance qualification

Custom cell mimics represent an emerging solution for addressing the challenge of biological reference material inconsistency. These precision-engineered tools replicate key phenotypic and functional characteristics of target cell types, enabling earlier potency assay development before final clinical material is available. They facilitate direct MoA-based functional readouts while providing standardization through stable, lot-matched materials that reduce variability across runs, sites, and operators [27].

Regulatory Strategy and Global Development Considerations

Navigating the divergent expectations of the FDA and EMA requires a proactive, strategically planned approach to potency assay development and validation. The following diagram outlines an integrated regulatory strategy for global cell therapy development.

Strategic Implementation Guidelines

Early Regulatory Engagement: Conduct parallel consultations with FDA and EMA during early development stages to identify potential divergences in potency expectations. Utilize parallel scientific advice procedures when significant differences emerge [9] [28].

Phase-Appropriate Assay Development: Implement a risk-based approach that focuses development resources on the most critical potency parameters early, while maintaining flexibility for assay refinement. Recognize that full validation may not be feasible until later stages, but MoA-relevance should be established early [22].

Expedited Pathway Utilization: Strategically leverage expedited programs (FDA RMAT, EMA PRIME) while recognizing that these pathways may have distinct potency data requirements. The FDA's expedited programs generally result in faster approval timelines compared to EMA standard procedures [9].

Integrated Control Strategy: Develop a comprehensive potency assurance strategy that integrates with existing quality systems rather than creating separate documentation structures. This approach reduces sponsor burden while maintaining regulatory compliance [22].

The successful development and regulatory approval of cell therapies depends fundamentally on robust potency assays that accurately reflect the product's Mechanism of Action. While EMA and FDA maintain distinct regulatory frameworks with differing emphases on data requirements and review timelines, both agencies converge on the essential principle that potency measurements must be biologically relevant and quantitatively reliable. By implementing strategic, phase-appropriate potency assay development aligned with therapeutic MoA, developers can navigate regulatory complexities, accelerate global approval timelines, and ultimately deliver consistently effective therapies to patients. The evolving regulatory landscape continues to emphasize potency as the critical link between product quality and clinical performance, making MoA-driven assay design an indispensable component of successful cell therapy development.

For developers of Cell Therapy Products (CTPs), demonstrating product potency is a fundamental regulatory requirement for market approval in both the United States (US) and the European Union (EU). Potency is defined as the quantitative measure of the biological activity of a product, which should be linked to its relevant biological properties and, ideally, its clinical performance [1]. The inherent complexity of cell therapies—viable products with multifaceted mechanisms of action (MoA)—makes potency testing particularly challenging. A single, monolithic assay is often insufficient to capture the full spectrum of biological activity. Consequently, a "matrix approach," which employs a combination of complementary test methods, has emerged as a scientifically rigorous and regulatory-accepted strategy to address this complexity [3] [1].

This approach is critical for navigating the distinct regulatory frameworks of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). While both agencies acknowledge the necessity of the matrix approach, their expectations and specific requirements exhibit key nuances that sponsors must incorporate into their global development strategies [12]. This guide provides an objective comparison of these regulatory stances, supported by experimental data and detailed protocols, to inform the strategies of researchers, scientists, and drug development professionals.

Regulatory Comparison: EMA vs. FDA Expectations

A clear understanding of the regulatory landscape is essential for designing an effective potency testing strategy. The following table summarizes the core positions of the two agencies.

Table 1: Comparison of EMA and FDA Potency Testing Requirements for Cell Therapies

Aspect	U.S. Food and Drug Administration (FDA)	European Medicines Agency (EMA)
Core Requirement	A validated, quantitative functional potency assay is expected for product release for pivotal trials and commercial supply [1] [12].	A functional assay is required, but validated surrogate assays can be used for release if correlated with a functional characterisation assay [1].
Assay Validation	Method validation according to ICH Q2(R2) is expected for the release assay [29].	For release testing, validated methods are expected; for characterisation, qualified methods are acceptable [1].
The "Matrix Approach"	Recognized as necessary. Analysis of 31 approved CTPs shows an average of 3.4 potency tests per product [3].	Implicitly supported through the acceptance of multiple methods to address complex MoAs [1].
Common Potency Test Types	"Viability and Count" (61% of products) and "Expression" (65% of products) are most frequently used, often together [3].	Similar test types are used, with an emphasis on assays reflecting the biological effect.
Potency for Viral Vectors (for in vitro use)	Classified as a drug substance; an enhanced control approach with functional assays is expected [12].	Considered a starting material; infectivity and transgene expression may be sufficient, especially in early development [12].

A review of 31 FDA-approved CTPs reveals the practical application of the matrix approach. These products employ a combined total of 104 potency tests, with an average of 3.4 tests per product. The most common test categories are "Viability and Count" (used by 61% of CTPs) and "Expression" (used by 65% of CTPs), and these two types are most frequently used in combination for the same product [3]. This data underscores that regulators routinely accept a multi-faceted testing strategy to assure product quality.

Table 2: Distribution of Potency Test Types for 31 FDA-Approved CTPs [3]

Potency Test Category	Description	Percentage of Non-Redacted Tests
Viability and Count	Measurements of cell viability, proliferation, or total cell count.	52%
Expression	Detection of specific gene or protein expression (e.g., CAR expression by flow cytometry).	27%
Bioassays	Functional assays measuring a biological response (e.g., cytotoxicity).	7%
Genetic Modification	Tests for vector copy number, transduction efficiency, etc.	9%
Histology	Microscopic evaluation of tissue structure.	3%

Experimental Data: Implementing the Matrix Approach

Case Study: ProtheraCytes CD34+ Cell Therapy

A prime example of the matrix approach is the development and validation of a potency assay for ProtheraCytes, an expanded autologous CD34+ cell therapy for acute myocardial infarction. The proposed mechanism of action is cardiac regeneration through revascularization, mediated by the secretion of pro-angiogenic factors like Vascular Endothelial Growth Factor (VEGF) [29].

Experimental Objective: To develop a quantitative, validated potency assay that reflects the product's MoA for lot release.

Methodology and Protocol:

• Technology: An automated microfluidic immunoassay system (ELLA, Bio-Techne) using a VEGF-specific cartridge.
• Sample Preparation: Cell culture supernatants are collected from expanded CD34+ cells after a 9-day culture period. The supernatant is used directly in the assay without dilution.
• Assay Principle: The method is a fully automated sandwich ELISA. The system measures relative fluorescence units (RFU) proportional to VEGF-antibody complexes and reports concentration against a factory-calibrated standard curve.
• Validation Parameters: The assay was validated per ICH Q2(R2) guidelines for the following [29]:
  • Specificity: Demonstrated no interference from the culture medium (StemFeed).
  • Linearity and Range: Confirmed from 20 pg/mL to 2800 pg/mL (R² = 0.9972).
  • Accuracy: Mean recovery rates between 85% and 105%.
  • Precision: Repeatability precision (CV ≤ 10%) and intermediate precision (CV ≤ 20%).

Supporting Data and Matrix Context: In this case, VEGF secretion serves as the primary functional potency assay. However, to fully characterize the product, this is likely part of a larger matrix that includes other tests, such as:

• Viability and Count: Total number of viable CD34+ cells.
• Expression/Phenotype: Flow cytometry to confirm CD34+ cell identity.
• Bioassay (Potential): A complementary endothelial tube formation assay in vitro could further corroborate the angiogenic potency.

This validated, quantitative potency assay was deemed acceptable by multiple regulatory agencies, supporting its use for the release of clinical batches [29].

The Scientist's Toolkit: Key Research Reagent Solutions

Implementing a robust potency testing strategy requires specific tools and reagents. The table below details essential materials used in the field, with examples from the featured case study.

Table 3: Research Reagent Solutions for Cell Therapy Potency Assays

Reagent / Solution	Function in Potency Testing	Example from ProtheraCytes Case
Automated Immunoassay System	Provides quantitative, high-precision quantification of soluble analytes (e.g., cytokines) with minimal handling and high reproducibility.	ELLA system (Bio-Techne) with VEGF cartridge [29].
Cell Culture Medium	Supports the expansion and function of cells during manufacturing and potentially during the potency assay itself.	StemFeed medium [29].
Flow Cytometry Reagents	Antibodies and dyes used to measure cell surface and intracellular markers for identity, purity, and transgene expression.	Not specified in the case, but essential for "Expression" tests (e.g., anti-CAR antibodies) [3].
Reference Standards	A well-characterized material used as a comparator to ensure the consistency and accuracy of the potency assay over time.	Factory-calibrated VEGF standard curve; high and low control samples [29].
Cell-Based Assay Reagents	Components for functional bioassays, such as target cells, co-culture media, and detection reagents for cytotoxicity or other mechanisms.	Not used in the primary VEGF assay, but would be needed for a cytotoxicity bioassay for a CAR-T product [1].

Visualizing the Pathways and Workflows

Regulatory Decision Pathway for Potency Assays

The following diagram outlines the logical decision process for selecting a potency testing strategy based on the regulatory context, highlighting the divergent paths for EMA and FDA.

Experimental Workflow for VEGF Potency Assay

This workflow details the specific experimental steps used in the ProtheraCytes potency assay validation.

The "matrix approach" to potency testing is not merely a best practice but a necessity for the successful development of complex cell therapies. The experimental data and regulatory comparison clearly demonstrate that a single assay is rarely adequate. A strategic combination of tests—spanning viability, identity, and, crucially, function—is required to convincingly demonstrate that a product will deliver its intended clinical effect.

The primary differentiator between the EMA and FDA remains the strict FDA requirement for a validated functional assay for product release, whereas the EMA offers a pragmatic alternative pathway using correlated surrogate methods. For global developers, this means that designing a potency strategy that meets FDA expectations from the outset provides the most efficient path to worldwide approval. As the regulatory landscape evolves, with a new ICH Q5E annex for CGT comparability in development, further harmonization may be on the horizon [12]. However, for now, a well-designed, scientifically justified matrix approach is the most robust strategy for addressing the multi-faceted biological functions of cell therapies and satisfying global regulatory requirements.

For CAR-T cell therapies, potency assays are critical quality control tests required by regulatory agencies to ensure that each manufactured product batch can elicit its intended biological effect [14]. These assays demonstrate a direct link between the product's mechanism of action (MoA) and its clinical efficacy [14]. Among the various approaches used for approved Cell Therapy Products (CTPs), bioassays that measure functional activity are particularly important, though their usage is reportedly less frequent compared to simpler viability or expression tests [14].

This guide focuses on two cornerstone functional potency assays: the cytokine release assay (CRA), which measures T-cell activation, and various cytotoxicity assays, which measure direct tumor-killing ability. We will compare these methods, provide experimental protocols, and analyze them within the context of evolving regulatory expectations from the FDA and EMA.

Comparative Analysis of Cytokine Release and Cytotoxicity Assays

The table below summarizes the core characteristics of the primary potency assays used for CAR-T cell products.

Table 1: Comparison of Key CAR-T Cell Potency Assays

Assay Type	Measured Parameter	Principle	Throughput	Key Advantages	Key Limitations
Cytokine Release (CRA)	T-cell activation (e.g., IFN-γ)	Quantification of cytokine secretion after antigen-specific stimulation [14]	High	Measures a critical MoA; can be highly specific	May not directly correlate with cytotoxic potency
Cytotoxicity (Chromium Release)	Target cell lysis	Measurement of radioactive (^{51})Cr released from lysed target cells [30]	Low	Considered a "gold standard"; highly sensitive	Uses radioactivity; endpoint measurement only [30]
Cytotoxicity (Bioluminescence)	Target cell viability	Quantification of luciferase activity in viable target cells [30]	High	High sensitivity; non-radioactive; allows for longitudinal measurement	Requires genetic modification of target cells [30]
Cytotoxicity (Impedance)	Target cell adherence	Measurement of electrical impedance as a proxy for adherent target cell health [30]	High	Label-free; real-time kinetic data	Cannot measure differential killing in heterogeneous cultures [30]
Cytotoxicity (Flow Cytometry)	Target cell lysis	Discrimination of live/dead target cells via fluorescent staining [30]	High	Multiplexing capability (phenotype); can use heterogenous targets; non-radioactive [30]	Complex data analysis; higher cost

Experimental Protocols for Key Potency Assays

Cytokine Release Assay (CRA) Protocol

The CRA measures T-cell activation, a critical step in the CAR-T cell mechanism of action.

• Step 1: Co-culture Setup. Seed CD19-expressing target cells (e.g., NALM-6 for ALL) in a multi-well plate. Add CAR-T cells at a specified Effector to Target (E:T) ratio (e.g., 1:1, 1:2) [30]. Include controls: CAR-T cells alone (background) and target cells alone.
• Step 2: Stimulation and Incubation. Incubate the co-culture for 18-24 hours at 37°C, 5% CO~2~ to allow for antigen-specific activation and cytokine secretion [14].
• Step 3: Supernatant Collection. Centrifuge the plate and carefully transfer the supernatant to a new plate, avoiding cell carry-over.
• Step 4: Cytokine Quantification. Use a sensitive immunoassay, such as an ELISA or electrochemiluminescence (ECL) assay, to quantify the concentration of IFN-γ or other relevant cytokines (e.g., IL-2) in the supernatant [14].

Cytotoxicity Assay via Flow Cytometry Protocol

This protocol provides a sensitive, non-radioactive method to quantify cell-mediated killing [30].

• Step 1: Target Cell Labeling. Label target cells (e.g., CD19+ tumor cell lines) with a fluorescent cell tracker dye, such as CellTrace Violet or CFSE [30] [31].
• Step 2: Co-culture Establishment. Co-culture the labeled target cells with CAR-T effector cells at various E:T ratios (e.g., from 50:1 to 1:1) for a defined period (e.g., 4-24 hours) [30].
• Step 3: Viability Staining. After incubation, stain the cells with a viability dye, such as 7-AAD or propidium iodide, which is excluded by live cells but penetrates the compromised membranes of dead cells [30] [32].
• Step 4: Flow Cytometry Acquisition and Analysis. Acquire samples on a flow cytometer. The percentage of specific lysis is calculated based on the proportion of stained (dead) cells within the fluorescently labeled target cell population. The assay can be validated for precision, with intra-assay precision demonstrated via technical duplicates [32].

Diagram 1: Cytotoxicity Assay Workflow.

The Scientist's Toolkit: Essential Reagents and Materials

Successful execution of potency assays requires carefully selected and validated reagents.

Table 2: Essential Research Reagents for CAR-T Potency Assays

Reagent/Material	Function	Example Specifics
Anti-CD19 CAR Detection Reagent	Flow cytometric identification and quantification of CAR-positive T cells [32]	Biotinylated CD19 antigen followed by fluorochrome-conjugated anti-biotin antibody [32]
Cell Trace Dyes (CFSE, CellTrace Violet)	Fluorescent, stable labeling of target cells for discrimination in co-culture [30]	Succinimidyl ester (SE) dyes that covalently bind intracellular amines [30]
Viability Dyes (7-AAD, Propidium Iodide)	Discrimination of live/dead cells by flow cytometry; penetrate compromised membranes of dead cells [30] [32]	7-AAD is a common nucleic acid dye used in flow cytometry [32]
Cytokine-Specific Antibodies	Capture and detection of secreted cytokines (e.g., IFN-γ) in CRA [14]	Used in ELISA or ECL-based multiplex immunoassays
CD19+ Target Cell Lines	Provide antigen-specific stimulus for CAR-T cell activation and killing	Lines such as NALM-6 (ALL) or other CD19-expressing B-cell malignancies
Lucent Target Cell Lines	Enable bioluminescence-based cytotoxicity assays; express luciferase [30]	Requires genetic modification of target cells [30]

Regulatory Perspectives: FDA and EMA Requirements

A comparative analysis of approved Cell Therapy Products (CTPs) reveals trends in regulatory acceptance of potency assays. For the 31 US FDA-approved CTPs, the average number of potency tests per product is 3.4 [14]. While "Viability and count" (52%) and "Expression" (27%) are the most commonly used tests, functional bioassays are critically important [14].

• FDA Perspective: The FDA requires a potency test for the release of all biologics, including CAR-T products [14]. The agency acknowledges the complexity and often employs a "weight-of-evidence" approach, where multiple assays collectively demonstrate potency [14]. For example, approved CAR-T products like Kymriah (tisagenlecleucel) have their potency assessed through a combination of CAR expression (by flow cytometry) and a functional CRA (IFN-γ release) [14].
• EMA Perspective: The new EMA guideline on investigational Advanced Therapy Medicinal Products (ATMPs), effective July 2025, emphasizes a risk-based approach to development [20]. It encourages sponsors to proactively plan for robust data collection and highlights that "immature quality development may compromise use of clinical trial data to support a marketing authorization" [20]. While significant regulatory convergence has been achieved between the FDA and EMA, particularly in Chemistry, Manufacturing, and Controls (CMC), differences remain in areas such as donor eligibility determination and Good Manufacturing Practice (GMP) compliance expectations [20].

Cytokine release and cytotoxicity assays are indispensable tools for establishing the potency of CAR-T cell therapies. The CRA directly measures a key MoA—T-cell activation—while cytotoxicity assays quantify the ultimate effector function. The choice of specific cytotoxicity method involves a trade-off between throughput, sensitivity, and technical complexity.

The regulatory landscape is dynamic. Both the FDA and EMA emphasize the importance of robust, phase-appropriate potency assays. While convergence is evident, developers must navigate nuanced differences. Future directions include the adoption of more predictive and sensitive methods, such as liquid biopsy-based monitoring (e.g., the HDSCA-HemeCAR workflow) that can simultaneously track persisting CAR-T cells and malignant B cells with high sensitivity [31], and the development of "armored" CAR-T cells engineered to secrete cytokines like IL-18 to enhance potency [33]. A deep understanding of both the technical aspects of these assays and the evolving regulatory expectations is crucial for the successful development and approval of next-generation CAR-T cell therapies.

Tumor-infiltrating lymphocyte (TIL) therapy represents a groundbreaking advancement in cancer immunotherapy, harnessing the body's own immune cells to combat malignancies. This personalized treatment approach involves extracting T cells that have naturally infiltrated a patient's tumor, expanding them exponentially in laboratory conditions, and reinfusing them to mount a potent anti-tumor response [34] [35]. The recent FDA approval of lifileucel (Amtagvi) in February 2024 marked a historic milestone as the first cellular therapy approved for a solid tumor, specifically for advanced melanoma [34]. This approval demonstrated remarkable efficacy, with a pivotal trial showing 31.5% of melanoma patients experiencing tumor reduction and 43.5% maintaining remission for over twelve months despite previous treatment failures [34].

However, the development and commercialization of autologous TIL therapies present extraordinary challenges that span manufacturing complexities, regulatory hurdles, and logistical obstacles. Unlike conventional pharmaceuticals, each TIL therapy lot constitutes a unique product derived from an individual patient, introducing inherent variability that complicates both manufacturing and regulatory assessment [34] [36]. The successful navigation of these challenges requires innovative approaches to potency testing, manufacturing optimization, and regulatory strategy, particularly when addressing divergent requirements between the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) [9].

TIL Therapy Manufacturing: Technical Complexities and Innovative Solutions

The TIL Manufacturing Process

The journey of TIL therapy from tumor resection to patient reinfusion is a complex, multi-stage process requiring precise execution at each step. The standard protocol begins with surgical resection of tumor tissue, followed by enzymatic digestion to extract the population of tumor-infiltrating lymphocytes [34] [35]. These extracted TILs are then cultivated in laboratory conditions with interleukin-2 (IL-2), allowing them to multiply into billions of cells over several weeks [34]. Before reinfusion, patients undergo lymphodepleting chemotherapy to create space for the expanded TILs, followed by IL-2 administration to stimulate further TIL growth and activation after reinfusion [34] [35].

Critical Manufacturing Challenges

Donor-to-Donor Variability: The cellular starting material derived from patient tumors demonstrates significant biological variability, representing the single largest source of variability in the production process [36]. This heterogeneity manifests in differences in T cell viability, proliferation capacity, and phenotypic composition, directly impacting manufacturing success rates and final product characteristics [36].

Manufacturing Timeline and Cell Stemness: Traditional TIL manufacturing processes required extensive culture periods, often resulting in T cell exhaustion and diminished therapeutic potential. Maintaining T cell "stemness" – the capacity for self-renewal and persistence – is now recognized as crucial for clinical efficacy [34]. Shortened culture duration correlates with longer telomere length, improved stemness, better persistence, and positive clinical outcomes [34].

Scale-Up and Contamination Risks: Traditional open-system culture methods required extensive operator interventions, increasing contamination risks and demanding highly skilled personnel [34]. Furthermore, final product volumes for TIL therapies often reach 30-50 liters, necessitating specialized equipment and dedicated facilities [34].

Manufacturing Innovations

Recent breakthroughs in TIL manufacturing have addressed these challenges through technological innovations:

Young TILs and Rapid Expansion Protocols: The development of "Young TILs" in 2008 marked a critical turning point, rapidly expanding TILs without lengthy tumor recognition screening, reducing manufacturing time to approximately 20 days [34]. Young TILs exhibit superior characteristics, including higher antigen reactivity, longer telomeres, and increased CD27 and CD28 expression – all factors associated with improved proliferative capacity and persistence after infusion [34].

Advanced Bioreactor Systems: To address contamination risks and scale-up challenges, gas-permeable G-Rex flasks have emerged as game-changing tools, supporting large media volumes without compromising gas exchange and enabling 2310-fold cell expansion compared to 729-fold with traditional methods [34]. Additionally, the WAVE bioreactor offers a closed-system alternative that produces comparable cell numbers in minimal volumes with reduced manipulation requirements [34].

Process Optimization: The Epi-R P2 protocol represents another manufacturing breakthrough, reducing production time to less than three weeks while maintaining cellular quality and yielding approximately 60 billion T cells [34]. This shortened protocol preserves stem-like qualities essential for clinical efficacy, with CD39 and CD69 double-negative memory-progenitor stem-like TILs strongly correlating with in vivo persistence and complete cancer regression [34].

Potency Testing: Navigating FDA and EMA Regulatory Frameworks

The Critical Role of Potency Assessment

Potency testing represents a cornerstone of regulatory evaluation for cell-based immunotherapies, serving as a essential quality attribute that demonstrates a product's biological activity and predictive therapeutic effect [19]. For autologous TIL therapies like lifileucel, potency assessment is particularly challenging due to product heterogeneity, complex mechanisms of action, and the absence of standardized assays across the industry [19] [18]. Both the FDA and EMA require rigorous potency testing, but their approaches reflect distinct regulatory philosophies and technical expectations that developers must navigate for global approval.

Comparative Analysis of FDA and EMA Requirements

Table 1: Key Differences in FDA and EMA Regulatory Approaches to Potency Testing for Cell Therapies

Aspect	FDA (U.S. Food and Drug Administration)	EMA (European Medicines Agency)
Overarching Guideline	Potency Assurance for Cellular and Gene Therapy Products (Draft Guidance, 2023) [11]	Guideline on potency testing of cell-based immunotherapy medicinal products for the treatment of cancer (CHMP/BWP/271475/06) [18]
General Approach	Flexible, case-by-case approach allowing for various assay strategies [11]	Structured framework with defined expectations for assay validation [18]
Potency Assay Strategy	Accepts composite assays and surrogate markers; emphasizes link to clinical mechanism [11]	Prefers direct measurement of biological activity; requires multi-parameter assessment [19]
Method Validation	Focus on assay robustness and reproducibility [11]	Detailed requirements for specificity, accuracy, precision, and limits of detection [18]
Stability Studies	Requires real-time stability data supporting potency claims [11]	Similar requirements with emphasis on potency retention throughout shelf-life [18]

Strategic Approach to Divergent Requirements

The divergent regulatory expectations between agencies necessitate strategic planning for global development programs:

Early Regulatory Engagement: Sponsors should engage with both agencies early through FDA Type B meetings and EMA Scientific Advice to anticipate differences in potency testing requirements [9]. This proactive approach allows for the development of assay strategies that satisfy both agencies' expectations, potentially avoiding costly delays and revalidation efforts.

Robust Assay Design: Developing potency methods that capture multiple mechanisms of action provides the strongest foundation for regulatory approval across jurisdictions [19]. For TIL therapies, this typically includes assessment of:

• Tumor cell killing capacity (cytotoxicity)
• Antigen-specific recognition and activation
• Cytokine production profile
• T cell phenotype and differentiation status [19]

Comparative Potency Testing: Implementing a comprehensive potency testing framework requires meticulous experimental design and execution. The following methodology outlines key approaches for TIL therapy assessment:

Table 2: Experimental Approaches for TIL Potency Testing

Assay Type	Methodology	Key Parameters Measured	Regulatory Applicability
Cytotoxicity Assay	Co-culture of TILs with tumor target cells; measurement of target cell death via flow cytometry or luminescence	Percentage specific lysis; lytic units per million cells	FDA & EMA (Direct biological activity)
Cytokine Release Assay	TIL stimulation with tumor antigens or nonspecific activators; multiplex cytokine analysis	IFN-γ, IL-2, TNF-α production; activation markers	FDA & EMA (Functional response)
Phenotypic Characterization	Multicolor flow cytometry for T cell surface and intracellular markers	Memory/effector subsets (CD45RA, CCR7, CD62L); exhaustion markers (PD-1, LAG-3, TIM-3)	FDA & EMA (Product characterization)
Proliferation Capacity	CFSE dilution or thymidine incorporation after antigen stimulation	Division index; precursor frequency	Primarily FDA (Surrogate for persistence)

Analytical Framework: TILScout and Computational Approaches

Deep Learning for TIL Assessment

Recent advances in computational pathology have enabled more sophisticated assessment of TIL potency and characteristics. TILScout, a pan-cancer deep-learning approach, computes patch-level TIL scores from whole slide images (WSIs) with remarkable accuracy [37]. This methodology achieved accuracies of 0.9787 and 0.9628, and AUCs of 0.9988 and 0.9934 in classifying WSI patches into TIL-positive, TIL-negative, and other/necrotic categories on validation and independent test sets, respectively, surpassing previous studies [37].

Experimental Workflow for TIL Analysis

The following diagram illustrates the comprehensive experimental workflow for TIL analysis and potency assessment, integrating both laboratory and computational approaches:

Research Reagent Solutions for TIL Analysis

Table 3: Essential Research Reagents for TIL Therapy Development

Reagent Category	Specific Examples	Function in TIL Development
Cell Culture Media	Serum-free media with IL-2	Supports TIL expansion while maintaining functionality and reducing contamination risk [34]
Activation Reagents	Anti-CD3 antibody, IL-2	Activates T cells and promotes proliferation during manufacturing [36]
Phenotypic Markers	Anti-CD3, CD4, CD8, CD45RA, CCR7, PD-1 antibodies	Characterizes T cell subsets and differentiation status [34]
Cytokine Detection	IFN-γ, IL-2, TNF-α ELISA or Luminex	Measures functional potency through cytokine secretion profile [19]
Vector Systems	Retroviral vectors for genetic modification	Enables T cell engineering for enhanced functionality [36]

Global Regulatory Navigation: Case Examples and Strategic Insights

Clinical Evidence Requirements

The divergent approaches between FDA and EMA extend to clinical evidence requirements for TIL therapies. A recent study published in JAMA Internal Medicine found that only 20% of clinical trial data submitted to both agencies matched, revealing major inconsistencies in regulatory expectations [9]. These discrepancies manifest particularly in:

Trial Design Endpoints: The FDA often accepts real-world evidence and surrogate endpoints for accelerated approval, while the EMA typically requires more comprehensive clinical data with emphasis on larger patient populations and long-term efficacy [9].

Approval Timelines: The FDA's expedited pathways (RMAT, Fast Track, Breakthrough Therapy) allow earlier market access, while EMA's PRIME and Conditional Marketing Authorization pathways, though similar in intent, often involve more extensive data requirements and longer review periods [9].

Successful Regulatory Strategy Components

Analysis of successful TIL therapy development programs reveals several key strategic components:

Proactive Regulatory Engagement: Companies that engaged early with both agencies through formal scientific advice procedures were better positioned to address divergent requirements before initiating pivotal studies [9] [38]. This includes anticipating differences in potency testing methodologies, stability data requirements, and clinical endpoint definitions.

Harmonized Clinical Development Plans: While complete harmonization between FDA and EMA requirements may be unattainable, developing a core clinical protocol with region-specific adaptations represents a balanced approach [9]. This strategy includes:

• Adaptive trial designs that accommodate both agencies' statistical expectations
• Collection of additional biomarker data to address specific regulatory concerns
• Strategic planning for regional patient recruitment to meet geographic requirements [39]

Robust Post-Marketing Commitments: Both agencies mandate extensive post-approval monitoring for cell therapies, though with different emphases. The FDA requires 15+ years of long-term follow-up (LTFU) for gene therapies, while EMA enforces a decentralized pharmacovigilance system with country-specific compliance requirements [9]. Implementing a comprehensive pharmacovigilance system that addresses both frameworks is essential for global commercialization.

The development of autologous TIL therapies like lifileucel represents a paradigm shift in cancer treatment, offering new hope for patients with advanced solid tumors. However, the path to successful commercialization requires navigating complex manufacturing, analytical, and regulatory challenges. The divergent requirements between FDA and EMA, particularly in potency testing and clinical evidence generation, necessitate sophisticated regulatory strategies that begin early in development.

Future success in the TIL therapy landscape will depend on continued innovation in manufacturing technologies to enhance product consistency, the development of more predictive potency assays that satisfy global regulatory standards, and strategic regulatory planning that anticipates and addresses regional differences. As the field evolves toward more genetically modified TIL products and combination approaches, the regulatory frameworks will similarly advance, requiring ongoing engagement and adaptation from developers.

The case of lifileucel demonstrates that despite the significant challenges, coordinated efforts across technical, clinical, and regulatory domains can successfully bring transformative TIL therapies to patients while meeting the rigorous standards of multiple global regulatory agencies. This achievement establishes a foundation for the next generation of TIL therapies that will potentially expand to additional cancer indications and benefit broader patient populations.

Selection and Qualification of Critical Reagents and Reference Materials

The development of cell and gene therapies (CGT), known as Advanced Therapy Medicinal Products (ATMPs) in the European Union, presents unique challenges in potency assessment. Potency—defined as the quantitative measure of a product's biological activity—is considered a Critical Quality Attribute (CQA) by regulatory agencies worldwide [1] [4]. For cell therapies, potency testing is particularly complex because these products are often viable entities with multifaceted mechanisms of action (MoA) that cannot be fully characterized by single-attribute testing [1].

The selection and qualification of critical reagents and reference materials used in potency assays represents a fundamental aspect of cell therapy development. These materials form the foundation of the analytical methods that demonstrate product consistency, manufacturing control, and ultimately, ensure that patients receive a therapy with predictable biological activity [4] [27]. The regulatory expectations for these materials vary between the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), creating a complex landscape for developers seeking global approval [20] [40].

Regulatory Framework Comparison: EMA vs FDA

Foundational Guidelines and Approaches

While both agencies recognize potency as a critical quality attribute, their guidance documents and regulatory emphasis display notable differences in approach and specific requirements.

Table 1: Foundational Regulatory Guidelines for Potency Testing

Agency	Key Guideline Documents	Primary Terminology	Overall Approach
U.S. FDA	- Potency Assurance for Cellular and Gene Therapy Products (Draft, 2023) [11]- Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (2011) [11]	Cell and Gene Therapy Products (CGT) [40]	Requires a quantitative functional potency assay for product release; emphasizes mechanism-of-action [1].
EU EMA	- Guideline on human cell-based medicinal products (EMEA/CHMP/410869/2006) [18]- Guideline on quality, non-clinical and clinical requirements for investigational ATMPs (2025) [20]	Advanced Therapy Medicinal Products (ATMPs) [40]	Acknowledges challenges of functional assays; may allow validated surrogate assays for release if correlated with a functional characterization assay [1].

A nuanced but critical difference lies in the acceptance of surrogate methods. The FDA expects a functional potency assay for product release, whereas EMA guidance provides more flexibility, stating that "for release testing validated surrogate assays could be utilized, provided that a functional assay is available as a characterisation tool and that the assays correlate with each other" [1]. This distinction directly impacts the strategy for selecting and qualifying critical reagents, as the FDA's stance often necessitates more complex, functionally-oriented biological reagents.

Analytical Method and Validation Expectations

The requirements for assay validation and the use of reference materials also demonstrate regional variations that affect development strategies.

Table 2: Analytical Method and Validation Expectations

Aspect	FDA Expectation	EMA Expectation
Potency Assay for Release	A quantitative, validated, functional potency assay is required for product release [1].	A combination of methods may be used; validated surrogate assays are potentially acceptable for release with justification [1].
Reference Standards	Emphasis on well-characterized in-house reference standards for relative potency calculation [4].	Similar expectations for characterized standards, with alignment to Ph. Eur. monographs where relevant [18].
Method Validation Timing	Assays should be validated for pivotal trials and licensure; qualified methods are acceptable for early-phase studies [1].	"Validated methods are expected for release and stability testing... For early clinical development, qualified methods are generally accepted" [1].
Phase-Appropriateness	Acknowledged in principle, but industry feedback indicates a need for more clarity on phase-appropriate potency assurance [22].	Explicitly endorsed in the 2025 clinical-stage ATMP guideline, advocating a risk-based approach [20].

Practical Implementation: Assay Strategies and Reagent Selection

Analysis of Approved Product Potency Tests

An analysis of the 31 FDA-approved cell therapy products (CTPs) through 2024 provides concrete evidence of current industry practices and regulatory acceptance [3]. A total of 104 potency tests were reported for these products, averaging 3.4 tests per CTP (Standard Deviation: 2.0) [3]. This data underscores that a multi-attribute approach (a "potency matrix") is common for characterizing complex cell-based products.

Table 3: Potency Test Types for FDA-Approved Cell Therapy Products (2010-2024) [3]

Category of Potency Test	Frequency of Use	Examples
Viability and Count	37 tests (52% of non-redacted tests)	Cell viability, Total nucleated cell count, CD34+ cell count [3].
Expression	19 tests (27% of non-redacted tests)	CAR expression, Marker expression (e.g., CD73, CD90, CD105) [3].
Bioassays	7 tests (10% of non-redacted tests)	Cytotoxicity, IFN-γ release assay, Enzyme activity [3].
Genetic Modification	6 tests (8% of non-redacted tests)	Vector copy number, Transduction efficiency [3].
Histology	2 tests (3% of non-redacted tests)	Histological evaluation of tissue structure [3].

The high prevalence of "Viability and Count" and "Expression" tests indicates their utility as core, often surrogate, elements of a potency matrix. However, the presence of bioassays for 23% of products (with potential for more among redacted tests) confirms that functional assessment remains a vital component for many CTPs, aligning with regulatory expectations for mechanism-of-action-based testing [3].

Critical Reagents and Reference Materials Toolkit

The development and execution of the potency tests described above rely on a foundation of well-qualified critical reagents and reference materials.

Table 4: Research Reagent Solutions for Cell Therapy Potency Testing

Reagent Category	Function & Importance	Qualification Requirements
Cell-Based Reference Materials	- Primary Target Cells: Used in bioassays (e.g., cytotoxicity) to measure functional potency [1].- Custom Cell Mimics: Engineered cells providing consistent antigen presentation for CAR-T potency assays (e.g., IFN-γ release), offering superior lot-to-lot consistency [27].	- Identity, Purity, Viability, Functional Responsiveness, Stability [27].
Critical Antibodies & Flow Cytometry Reagents	- Phenotyping Panels: For "Expression" tests to quantify surface/intracellular markers (e.g., CAR expression) [3].- Detection Antibodies: For cytokine capture assays (e.g., ELISA, ELISpot) used as functional potency measures [4].	- Specificity, Sensitivity, Titration, Cross-reactivity, Clone Stability, Fluorochrome Brightness Index [4].
Reference Standards & Controls	- Product-Specific Reference Standard: A well-characterized batch of the product itself used as a comparator in relative potency assays [4].- Assay Controls: Positive/Negative controls to monitor assay performance and system suitability [4].	- Comprehensive Characterization (identity, purity, potency), Stability under storage conditions, Homogeneity [4].
Culture Reagents & Cytokines	- Cell Culture Media/Feeds: Maintain cell viability and function during extended bioassays.- Recombinant Cytokines/Growth Factors: Used to stimulate or differentiate cells in functional assays.	- Endotoxin Testing, Sterility, Growth-Promotion Testing, Performance Qualification [18].

Experimental Protocols for Qualification

Protocol: Qualification of a Custom Cell Mimic for a CAR-T Potency Assay

Objective: To qualify a custom cell mimic (e.g., TruCytes) as a critical reagent for use in a mechanism-of-action-based potency assay (e.g., IFN-γ release) for a CD19-targeting CAR-T product.

Methodology:

• Identity Confirmation: Verify the presence of the CD19 antigen on the cell mimic surface using flow cytometry with certified anti-CD19 antibodies. Acceptable criteria: ≥95% of cells are CD19 positive.
• Functional Potency Assay Performance:
  • Co-culture CAR-T cells with the qualified cell mimics at varying Effector:Target (E:T) ratios.
  • After a defined incubation period (e.g., 24 hours), measure IFN-γ secretion in the supernatant using a validated ELISA.
  • Parallel-Line Analysis: Generate a dose-response curve for the test sample (new CAR-T batch) and the reference standard. The relative potency is calculated by comparing the horizontal distance (on the log-scale) between the two parallel regression lines [4].
• Stability Monitoring: Perform real-time and accelerated stability studies to establish the shelf-life of the frozen cell mimics. Assess post-thaw viability, identity (CD19 expression), and functionality (ability to induce IFN-γ release from a control CAR-T batch) at predetermined time points.

The following workflow diagrams the development and qualification pathway for critical reagents used in cell therapy potency assays, from selection through to application in the control strategy.

Figure 1: Critical Reagent Qualification and Implementation Workflow

Protocol: Statistical Analysis for Relative Potency

Objective: To quantify the relative potency of a test sample against a reference standard using a cell-based bioassay, ensuring the analysis is robust and meets regulatory standards.

Methodology:

• Experimental Design: Test both the reference standard and the sample at a minimum of 3 concentrations, ideally in a dilution series spanning the expected linear range of the assay. Use multiple replicates (e.g., n=3) per concentration.
• Data Transformation: Convert raw response data (e.g., optical density, luminescence) as needed. Log transformation of the dose is standard for parallel-line analysis.
• Model Fitting - Parallel-Line Analysis:
  • Fit a linear regression model to the log-dose vs. response data for both the standard and the sample.
  • Test for parallelism (the fundamental assumption). This is typically done using an F-test to confirm there is no significant difference between the slopes of the two regression lines (e.g., p-value > 0.05 for the slope difference) [4].
  • If the parallelism assumption holds, the relative potency is calculated as the antilog of the horizontal distance between the two parallel lines: Potency = 10^(Δlog-dose).
• Report Results: The final result is expressed as a percentage relative to the standard (e.g., 98% relative potency) with associated confidence intervals, demonstrating the precision of the estimate.

The following diagram illustrates the logical decision process and statistical methodology for establishing a validated potency assay, highlighting the central role of the reference standard.

Figure 2: Potency Assay Validation and Analysis Decision Flow

The selection and qualification of critical reagents and reference materials is a foundational activity in building a robust, globally-compliant potency assay strategy for cell therapies. While the core scientific principles are shared between the FDA and EMA, key differences in regulatory philosophy—particularly regarding the strict requirement for a functional release assay versus the potential acceptance of correlated surrogate methods—directly impact resource allocation and development timelines.

A successful global strategy requires understanding these nuances, implementing a phase-appropriate approach early in development, and investing in well-characterized, stable critical reagents. The analysis of approved products confirms that a multi-faceted "potency matrix" is the norm, combining simpler, robust tests for routine release with more complex, functional assays for thorough product characterization. By prioritizing the qualification of critical reagents like cell-based reference materials and antibodies, developers can build a persuasive data package that demonstrates a deep understanding of their product's mechanism of action and ensures consistent biological activity for patients, thereby facilitating regulatory review across both major jurisdictions.

Navigating Differences in Starting Material Requirements (e.g., GMP-grade Plasmids)

The development of cell and gene therapies (CGTs) represents one of the most significant advances in modern medicine, offering potential cures for previously untreatable conditions. At the heart of this therapeutic revolution lies plasmid DNA (pDNA), a fundamental starting material used in the production of viral vectors, which are essential for delivering genetic material into patient cells [41] [42]. The global plasmid DNA manufacturing market, valued at $2.38 billion in 2024 and projected to reach $16.95 billion by 2034, reflects the critical importance of this biological component [41]. As more therapies progress through clinical development, regulatory scrutiny of starting materials has intensified, particularly regarding Good Manufacturing Practice (GMP) compliance and quality assurance. The divergent regulatory approaches between the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) toward these starting materials present significant challenges for developers seeking global approval [9] [12]. Understanding these differences is crucial for designing efficient manufacturing and control strategies that satisfy both regulatory regimes without compromising development timelines or product quality.

Comparative Analysis of FDA and EMA Regulatory Frameworks

Fundamental Differences in Starting Material Classification

The FDA and EMA employ fundamentally different conceptual frameworks for classifying and regulating plasmid DNA used in cell and gene therapy manufacturing, creating a complex regulatory landscape for developers [12].

• EMA Classification: The European Medicines Agency explicitly defines plasmid DNA as a 'starting material' – a biological entity that becomes an integral part of the final drug substance [12]. This classification carries significant regulatory implications, as it requires that these materials be manufactured in accordance with GMP principles throughout clinical development. The EMA's approach is rooted in the concept that quality must be built into the product from the very beginning of the manufacturing process, with the qualified person (QP) at the drug substance manufacturer bearing responsibility for ensuring starting material quality [12].

• FDA Classification: In contrast, the U.S. Food and Drug Administration does not formally define 'starting materials' in its regulatory lexicon [12]. Instead, the FDA typically categorizes plasmid DNA under the broader concept of 'critical raw materials' and expects manufacturers to implement a risk-based control strategy that becomes increasingly rigorous as products advance through clinical development phases. This approach offers greater flexibility during early-stage development but requires careful documentation and justification of control strategies [12].

Table 1: Fundamental Regulatory Classification Differences

Regulatory Aspect	FDA Approach	EMA Approach
Classification	Critical raw material	Starting material
GMP Requirements	Phase-appropriate, risk-based	Full GMP compliance
Manufacturer Oversight	Enhanced material control strategy	Qualified Person certification
Regulatory Basis	No formal definition of starting materials	Explicit definition in guidelines

Comparative Requirements for Key Manufacturing Aspects

The divergent classification philosophies between the two agencies translate into specific technical and regulatory requirements across multiple aspects of plasmid DNA manufacturing and control [12].

• Potency Assay Requirements: The FDA typically mandates validated functional potency assays to assess the biological activity of viral vectors produced using plasmid DNA, particularly for products entering pivotal clinical studies. The EMA, while recognizing the importance of potency assessment, may accept infectivity and transgene expression measurements as sufficient during early development phases, with a transition to more comprehensive potency assays as products approach marketing authorization [12].

• Viral Safety Testing: For replication competent virus (RCV) testing, the FDA requires testing at both the plasmid/vector stage and the final cell-based drug product level. The EMA considers that once RCV absence has been demonstrated in the viral vector, further testing of the genetically modified (GM) cells is generally not required [12].

• Donor Testing Requirements: While both agencies require comprehensive testing of human-derived starting materials, the EMA imposes additional requirements for autologous donations (where patients receive their own cells), whereas the FDA's requirements in this area are less extensive [12].

Table 2: Detailed Comparison of Technical Requirements

Technical Area	FDA Requirements	EMA Requirements
Potency Testing	Validated functional assays essential for pivotal studies	Infectivity and transgene expression may suffice in early phase
RCV Testing	Required for both vector and final cell product	Generally required only for vector stage
Allogeneic Donor Testing	Prescriptive requirements for screening and testing	Must comply with EU and member state laws
Autologous Donor Testing	Less extensive requirements	Additional testing requirements
Stability Data	Thorough assessment with real-time data for changes	Real-time data not always required for comparability

Experimental Data and Methodologies

Analytical Method Selection for Plasmid DNA Qualification

Robust analytical methods are essential for characterizing GMP-grade plasmid DNA and demonstrating compliance with regulatory requirements. The following methodologies represent current industry standards for plasmid DNA qualification [4].

• Vector Copy Number (VCN) Analysis: Quantitative polymerase chain reaction (qPCR) and digital droplet PCR (ddPCR) methods are employed to determine the number of plasmid copies per cell, a critical safety parameter. The experimental protocol involves: (1) extracting genomic DNA from transduced cells using column-based purification methods; (2) preparing standard curves with known plasmid copy numbers; (3) performing amplification with primers specific to the transgene and a reference genomic locus; (4) calculating VCN using the ΔΔCt method for qPCR or direct counting for ddPCR. Acceptance criteria typically specify ≤5 copies per cell to minimize oncogenic risk [4].

• Transduction Efficiency Assessment: Flow cytometry methods quantify the percentage of cells successfully transduced with the plasmid-derived vector. The standard protocol includes: (1) transducing target cells with the viral vector at varying multiplicities of infection (MOI); (2) incubating for 48-72 hours to allow transgene expression; (3) harvesting cells and staining with antibodies against the transgene product or using reporter systems (e.g., GFP); (4) analyzing using flow cytometry with appropriate isotype controls. This method provides critical data on vector functionality [4].

• Plasmid Identity and Integrity Verification: Next-generation sequencing (NGS) provides comprehensive characterization of plasmid identity and sequence integrity. The methodology involves: (1) fragmenting plasmid DNA via enzymatic or mechanical methods; (2) preparing sequencing libraries with platform-specific adapters; (3) performing high-throughput sequencing on platforms such as Illumina or Nanopore; (4) aligning sequences to reference plasmids to verify identity and detect sequence variants. This orthogonal method complements traditional restriction digest analysis [4].

Comparative Study Data: Regulatory Submission Analysis

A recent cross-sectional study comparing clinical evidence submitted to both agencies revealed significant disparities in regulatory requirements [43]. The investigation found that only 20% of clinical trial data submitted to both regulatory bodies matched, highlighting substantial differences in evidentiary expectations [9] [43]. This divergence creates considerable challenges for developers pursuing simultaneous approvals in both markets, often necessitating the collection of additional region-specific data or implementation of distinct analytical strategies.

Strategic Implementation Framework

Proactive Regulatory Planning

Successful navigation of the divergent FDA and EMA requirements demands strategic, early-stage planning with clearly defined implementation steps [9] [12].

• Early Regulatory Engagement: Pursue parallel scientific advice meetings with both agencies during preclinical development to identify potential divergences in requirements. Document all interactions and feedback thoroughly, and incorporate this intelligence into the overall development strategy. Early engagement allows developers to anticipate and address regulatory differences before committing to specific manufacturing or analytical approaches [9].

• Harmonized Quality Systems: Implement a unified quality system designed to meet the most stringent requirements of both agencies from the outset. This includes establishing comprehensive document control procedures, change management protocols, and deviation management systems that satisfy both FDA's Current Good Manufacturing Practice (cGMP) and EMA's GMP standards, creating a solid foundation for global regulatory submissions [12].

• Platform Process Development: Where scientifically justified, develop and validate platform manufacturing processes for plasmid DNA production that can be applied across multiple product candidates. Document process performance and product quality attributes extensively to build a data package that demonstrates consistency and control, which can be leveraged to support regulatory submissions in both jurisdictions [12].

Phase-Appropriate Control Strategy Implementation

Developing a phase-appropriate control strategy that evolves with the product's clinical progression is essential for managing regulatory expectations while maintaining development efficiency [4] [12].

• Preclinical Phase: Focus on developing fit-for-purpose analytical methods capable of detecting critical quality attributes (CQAs). Implement comprehensive plasmid characterization including identity, purity, potency, and safety attributes. Establish reference standards and preliminary specifications based on limited manufacturing experience and scientific rationale [4].

• Early Clinical Phase (Phase 1/2): Enhance method robustness and begin analytical method qualification. Implement statistical process control (SPC) for critical process parameters. Establish preliminary acceptance criteria based on manufacturing experience and clinical requirements. Begin formal stability studies under recommended storage conditions [4] [12].

• Late Clinical Phase (Phase 3): Complete full method validation for all release assays. Establish definitive acceptance criteria based on comprehensive analysis of manufacturing consistency and clinical experience. Finalize the control strategy with clearly defined in-process controls, release specifications, and characterization assays. Complete formal stability studies to support proposed shelf life [4] [12].

Essential Research Reagents and Solutions

The successful development and implementation of control strategies for GMP-grade plasmid DNA requires carefully selected reagents and materials designed to meet regulatory expectations [4] [44].

Table 3: Essential Research Reagent Solutions

Reagent Category	Specific Examples	Function in Plasmid DNA Qualification
Cell Lines	HEK293, HeLa, A549	Provide cellular systems for potency and infectivity assays; must be properly characterized and banked
qPCR/ddPCR Reagents	TaqMan assays, EvaGreen dye, droplet generation oil	Enable precise quantification of vector copy number and residual plasmid DNA
Reference Standards	Custom plasmid standards, cell line qualifications	Serve as assay controls for method qualification and validation
NGS Library Prep Kits	Illumina Nextera, Nanopore Ligation kits	Facilitate comprehensive plasmid sequence verification and identity testing
Flow Cytometry Reagents	Fluorescent antibodies, viability dyes, calibration beads	Allow quantification of transduction efficiency and transgene expression

Process Visualization

The following workflow diagram illustrates a comprehensive strategy for navigating the divergent FDA and EMA requirements for GMP-grade plasmid DNA.

Strategic Pathway for FDA and EMA Compliance

The divergent regulatory requirements for GMP-grade plasmid DNA between the FDA and EMA present significant challenges for global development of cell and gene therapies. These differences span classification frameworks, GMP expectations, potency testing requirements, and viral safety strategies [9] [12]. Success in this complex environment requires a proactive, strategically planned approach that incorporates early regulatory engagement, phase-appropriate control strategies, and thorough documentation practices. By understanding these regulatory distinctions and implementing harmonized yet flexible quality systems, developers can navigate the divergent requirements more efficiently, potentially accelerating global access to transformative cell and gene therapies for patients in need. As the regulatory landscape continues to evolve, with initiatives like the BioPhorum ATMP working group developing tools such as the Gene Therapy Potency Strategy Roadmap, sponsors should maintain ongoing vigilance regarding regulatory updates from both agencies [5].

Overcoming Common Hurdles: A Troubleshooting Guide for Potency Assay Development

Addressing High Variability and Robustness Issues in Bioassays

Regulatory Landscape: EMA vs. FDA Potency Testing Requirements

The regulatory environment for cell therapy potency testing is characterized by both convergence on fundamental principles and divergence in specific implementation timelines between the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Understanding these nuances is critical for developing robust, compliant bioassays.

Table 1: Key Regulatory Divergences Between FDA and EMA for Cell Therapy Potency Assays

Aspect	FDA Approach (2023/2024 Draft Guidance)	EMA Approach (2025 Guideline)
Core Principle	Comprehensive "potency assurance strategy" integrating Quality by Design (QbD) and Quality Risk Management (ICH Q9) [45].	General expectations for quality, safety, and efficacy, with specific guidelines for Advanced Therapy Medicinal Products (ATMPs) [5].
Phase-Appropriateness	Acknowledges phase-appropriateness, but potency assurance strategy expected from early clinical development [45] [10].	Explicitly states "an appropriate degree of method validation should be applied at each stage," with full validation for safety-relevant assays even in Phase 1 [5].
Assay Validation Timing	Method qualification is necessary prior to clinical investigation; full validation required for licensed products [45] [4].	All safety-relevant assays must be fully validated from Phase 1, creating a potentially higher initial burden [5].
Number of Assays	Recommends multiple release assays, with at least one bioassay measuring relevant biological activity [45].	Industry advocates for one robust in vitro potency assay addressing the main mechanism of action (MoA) for release, with others as characterization assays [10].
Clinical Hold Risk	Insufficient potency assurance can be a reason for clinical hold at any stage if it creates an unreasonable risk to patients [45] [10].	Variable interpretation by smaller national agencies relying on verbatim adoption of larger agency guidelines, complicating multi-regional trials [5].

A significant challenge is that regional agencies often adopt the guidelines of larger bodies verbatim, limiting the sponsor's flexibility to implement a single, global potency strategy [5]. This is particularly burdensome for therapies targeting ultra-rare diseases, where patient populations are spread across multiple regions [5]. The industry, through groups like the American Society of Gene & Cell Therapy (ASGCT), has advocated for more phase-appropriate flexibility, especially in early development where limited batch data and incomplete mechanistic understanding are common [10].

Root Causes of Bioassay Variability and Robustness Challenges

High variability in bioassays stems from multiple interconnected technical and biological factors. Understanding these root causes is the first step toward implementing effective mitigation strategies.

• Biological System inherent Variability: Cell-based potency assays are inherently variable due to factors such as cell passage number, metabolic activity, and receptor density [46]. Minor changes in culture conditions, media composition, or serum lot can significantly alter cell viability and drug response, leading to inconsistent results [46].

• Technical and Reagent Inconsistencies: A key source of variability is the lack of standardized, regulatory-ready reference materials. Many early-stage assays rely on research-grade cell lines or biological materials that lack the chain-of-custody documentation required for GMP compliance [27]. Inconsistencies in reagent stability, enzyme activity during cell detachment, and poor signal-to-noise ratios also contribute to a unreliable dynamic range [46].

• Operational and Transfer Issues: As assays move from R&D to QC and potentially between different sites, differences in operator technique, equipment calibration, and environmental conditions introduce variability. Without early harmonization of protocols, cross-site reproducibility becomes a major hurdle [46].

• Statistical and Analytical Limitations: Bioassays occasionally display unusually high variability that falls outside the assumptions of standard statistical analyses. This can be due to the assay's complexity or because it represents the best available option to capture a relevant biological process [47]. Applying inappropriate statistical models for dose-response curve fitting can further compromise data reliability [46] [48].

Strategic and Experimental Approaches for Robust Assay Development

Comprehensive Potency Assurance Strategy

A proactive, holistic strategy is more effective than focusing solely on the final release test. The FDA's recent draft guidance emphasizes building potency assurance into the entire product lifecycle [45].

Diagram 1: Potency Assurance Strategy Workflow

This strategy begins with defining a Quality Target Product Profile (QTPP) based on the product's mechanism of action (MoA) and intended clinical use [45]. Subsequently, potency-related Critical Quality Attributes (CQAs) are identified through prior knowledge, characterization studies, and nonclinical data [45]. A risk assessment is then conducted for each CQA, evaluating the impact of materials and critical process parameters (CPPs) [45]. Risks are mitigated through manufacturing process design and a comprehensive control strategy encompassing materials, in-process testing, and release testing [45]. Finally, this entire strategy must be reassessed and refined throughout the product development lifecycle as process and product understanding deepens [45].

Practical Experimental Protocols for Robustness

Implementing standardized experimental protocols is key to reducing variability. The following methodologies are critical for establishing control.

• Protocol 1: Standardized Cell Culture and Banking

  • Objective: To minimize variability originating from biological reagents.
  • Methodology: Establish a Master Cell Bank (MCB) under controlled conditions from a low-passage cell stock. rigorously characterize the cell bank for target antigen density, identity, and viability. Implement strict limits on cell passage number for assay use. Use standardized, qualified media and serum batches, and pre-test critical reagents like detection antibodies for optimal performance [46] [4].
  • Data Interpretation: Consistent performance within established parameters over at least 10 consecutive runs indicates a well-controlled cell system. A drift in signal or increased CV% suggests issues with cell health or reagent stability.

• Protocol 2: Design of Experiments (DoE) for Assay Optimization

  • Objective: To systematically identify and control critical assay parameters rather than using a one-factor-at-a-time (OFAT) approach.
  • Methodology: Select key parameters (e.g., cell density, incubation time with the ADC, detection reagent concentration). Use a statistical DoE model to study the main effects and interactions of these parameters. The output is measured against metrics like signal-to-background ratio, Z'-factor, and CV% [48].
  • Data Interpretation: An optimal assay window is achieved when the Z'-factor is >0.5. The model will identify which parameters most significantly impact the result, allowing for the definition of a robust operating range.

• Protocol 3: Implementation of Custom Reference Materials

  • Objective: To provide a consistent and qualified stimulus for the assay system, enabling earlier development and reducing variability.
  • Methodology: Utilize precision-engineered custom cell mimics (e.g., TruCytes). These are synthetic cells designed to replicate key phenotypic and functional characteristics of target cell types, such as specific surface markers. They can be engineered with tunable antigen density to standardize the activation strength delivered to effector cells like CAR-Ts [27].
  • Data Interpretation: The use of these stable, lot-matched materials reduces run-to-run and operator-to-operator variability. They enable direct MoA-based functional readouts (e.g., IFN-γ secretion) and provide a regulatory-friendly assay component, avoiding the challenges of qualifying in-house tumor cell lines [27].

Diagram 2: Assay Robustness Optimization Pathway

Advanced Statistical and Analytical Methods

When standard statistical methods assuming normal distribution are insufficient, robust statistical methods provide a more appropriate toolkit for both data analysis and assay optimization [47].

Table 2: Statistical Models for Potency Assay Data Analysis

Model	Application Context	Key Advantage	Consideration for Use
Parallel-Logistic (4PL/5PL)	Non-linear dose-response curves; most common for cell-based assays [4].	High precision for calculating relative potency; flexible for various curve shapes (4PL, 5PL).	Requires a sufficient number of data points to reliably fit the model; may be sensitive to outliers.
Parallel-Line Analysis	Linear portion of a dose-response curve [4].	Simpler model; robust and widely accepted for immunoassays.	Applicable only when the response is linear over the tested concentration range.
Slope-Ratio Analysis	Linear regressions where the slopes of the test and reference preparations are compared [4].	Useful for specific assay types where the parallel-line premise does not hold.	Less commonly applied than parallel-line or logistic models.
Robust Statistical Methods	Assays with unusually high variability that falls outside standard assumptions [47].	Provides more reliable estimates and confidence intervals when data contains outliers or is non-normal.	Requires specialized statistical expertise; impacts experimental design and interpretation [47].

The choice of model must be justified and appropriate for the type of response being measured. Furthermore, a key part of the control strategy is defining system suitability criteria and assay acceptance criteria based on the performance of validated reference standards [48].

The Scientist's Toolkit: Essential Reagent Solutions

Table 3: Key Research Reagent Solutions for Robust Potency Assays

Reagent / Material	Function in Potency Assay	Criticality for Robustness
Custom Cell Mimics (e.g., TruCytes)	Standardized, off-the-shelf target cells that replicate key patient cell characteristics for a consistent assay stimulus [27].	High: Replaces highly variable tumor cell lines or primary cells; enables earlier assay development with regulatory-ready materials.
GMP-Grade Cell Banks	Qualified Master and Working Cell Banks providing a consistent and traceable source of cells for the assay system.	High: Foundation of assay consistency; limits genetic drift and phenotypic changes that introduce variability.
Reference Standard	A well-characterized material used as a benchmark for calculating the relative potency of test samples.	Critical: Essential for system suitability and for ensuring the assay performs within a validated range across different runs and sites.
Qualified Critical Reagents	Key assay components like antibodies, cytokines, and detection substrates that have undergone rigorous pre-testing.	High: Lot-to-lot variability in these reagents is a major source of assay failure. Qualification ensures consistent performance.
Automated Liquid Handling Systems	Robotics for performing repetitive tasks like sample serial dilution and cell plating.	Medium-High: Reduces human error and operator-to-operator variability, significantly improving reproducibility [46].

Addressing high variability in cell therapy bioassays requires a multi-faceted approach that is deeply integrated with an evolving global regulatory landscape. Success hinges on moving beyond the potency assay as a mere QC test and embracing a holistic potency assurance strategy that is implemented in a phase-appropriate manner. By combining strategic planning rooted in QbD principles, rigorous experimental protocols using standardized reagents, and advanced statistical models, developers can build the robustness and regulatory confidence needed to accelerate the delivery of transformative cell therapies to patients.

Strategies for Managing Limited Sample Availability and Short Shelf-Lives

For developers of cell and gene therapies, limited sample availability and short product shelf-lives present significant challenges for potency testing, a critical quality attribute required by global regulatory agencies. These constraints create substantial technical and logistical hurdles in designing and executing potency assays that must demonstrate the specific biological activity of these complex living products [4] [1].

The inherent limitations of cell therapy products—including small batch sizes (particularly for autologous products), limited stability, and cellular heterogeneity—collide with regulatory expectations for comprehensive potency assessment [3] [10]. This article examines the divergent regulatory expectations between the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) and explores practical strategies and experimental approaches to address these challenges while maintaining regulatory compliance throughout the product development lifecycle.

Regulatory Landscape: EMA vs. FDA Perspectives

Fundamental Regulatory Framework Differences

While both agencies recognize the challenges of potency testing for cell therapies, their approaches and requirements display notable differences that developers must navigate:

Table: Key Regulatory Differences Between FDA and EMA for Cell Therapy Potency Testing

Aspect	U.S. FDA Approach	EMA Approach
Primary Potency Requirement	Quantitative functional potency assay for product release [1]	Allows validated surrogate assays for release when correlated with functional characterization assays [1]
Number of Potency Assays	Suggests multiple potency assays; industry advocates for one robust assay addressing main mechanism of action [10]	Acceptuses a combination of methods to address multiple functional mechanisms [1]
Phase Appropriateness	Acknowledged in guidance, but inconsistencies in application reported [10]	More flexible approach for early development; graduated requirements [1]
Validation Timing	Expected for pivotal clinical studies and commercial production [1]	Qualified methods generally accepted for early clinical development [1]

Analysis of Currently Approved Product Testing Strategies

An analysis of 31 FDA-approved cell therapy products reveals current industry practices that have successfully navigated regulatory requirements [3]:

• Average of 3.4 potency tests per product (standard deviation 2.0), with a range of 1-8 tests
• 52% of tests measure "Viability and count"
• 27% of tests measure "Expression" (gene or protein)
• Only 7% of tests are categorized as "Bioassays"
• 32% of potency tests are redacted in public documents, suggesting proprietary methods

This data indicates that while complex bioassays are less frequently used as primary release tests, successful regulatory strategies often employ a matrix approach combining multiple test types [3].

Strategic Approaches for Limited Samples & Short Shelf-Lives

Parallel Testing and Workflow Optimization

Conducting multiple required tests with limited sample material necessitates strategic experimental design and workflow optimization:

Figure 1: Parallel Testing Workflow for Limited Samples - This strategy divides limited samples for simultaneous testing, with rapid assays providing early decision points while longer functional assays proceed.

Tiered Potency Assay Development

A phased approach to potency assay development aligns with product development stage and addresses sample limitations:

Figure 2: Tiered Potency Assay Development - This approach progressively builds assay complexity as product knowledge increases and more samples become available, addressing limited early-stage resources.

Surrogate Assay Implementation

For products with particularly short shelf-lives, establishing validated surrogate assays that correlate with longer functional assays enables timely release decisions:

• EMA explicitly allows surrogate assays for release when correlation with functional characterization assays is demonstrated [1]
• FDA may accept surrogate approaches when justified by scientific rationale and correlation data
• Viability and phenotypic markers often serve as effective surrogates when correlated with functional mechanisms [3]

Case Study: VEGF Potency Assay for CD34+ Cell Therapy

Experimental Protocol and Methodology

A validated potency assay for ProtheraCytes (expanded autologous CD34+ cells) demonstrates practical implementation of strategies for limited samples [29]:

Table: Research Reagent Solutions for VEGF Potency Assay

Reagent/Equipment	Function in Assay	Key Features for Limited Samples
ELLA Automated Immunoassay System (Bio-Techne)	Automated VEGF quantification	Minimal handling, reduced variability, small sample volumes
Simple Plex VEGF-A Cartridge	Microfluidic VEGF detection	Specific antibody recognition, factory-calibrated standard curve
StemFeed Culture Medium	Cell expansion platform	Defined composition, supports CD34+ cell growth and VEGF secretion
VEGF-A Standard	Calibration and validation	Traceable reference for quantitative measurements

Sample Collection Protocol:

• Collect supernatant after 9 days of CD34+ cell expansion
• Centrifuge at 300 × g for 10 minutes to remove cellular debris
• Aliquot and freeze at -80°C if not testing immediately
• Thaw samples only once for analysis

Assay Execution:

• Load samples and controls into ELLA system
• Run automated immunoassay (approximately 90 minutes)
• System generates standard curve and calculates VEGF concentrations
• Report potency based on VEGF secretion per million cells

Validation Data and Performance Metrics

This approach successfully addressed sample limitation challenges while meeting regulatory expectations [29]:

• Linear range: 20 pg/mL - 2800 pg/mL (R² = 0.9972)
• Precision: CV ≤10% (repeatability), ≤20% (intermediate precision)
• Accuracy: Mean recoveries 85-105%
• Specificity: No interference from culture medium components
• Sample volume: Minimal requirements enabled by automated system

The assay successfully supported release of 38 clinical batches, demonstrating consistency with mean VEGF secretion of 596.2 ± 242.3 pg/mL for patient samples [29].

Implementation Framework and Best Practices

Strategic Potency Assay Development Pathway

Navigating the complex landscape of potency testing with sample constraints requires a systematic approach:

Figure 3: Strategic Potency Assay Development - This decision pathway incorporates critical assessment points for sample requirements and stability constraints throughout development.

Regulatory Engagement Strategy

Early and strategic regulatory engagement is critical for success:

• Seek parallel scientific advice from both FDA and EMA when possible to identify divergent expectations early [9]
• Leverage early phase flexibility to implement phase-appropriate strategies [10] [20]
• Document all development decisions regarding assay selection, especially those driven by sample limitations
• Present correlation data between different assay formats to support surrogate approaches

Successfully managing limited sample availability and short shelf-lives in cell therapy potency testing requires a multifaceted strategy that integrates scientific, technical, and regulatory approaches. The key lies in developing a phase-appropriate, risk-based potency assurance strategy that begins with simpler, rapid assays and evolves toward more complex, mechanism-reflective methods as product knowledge and sample availability increase.

By implementing parallel testing workflows, employing tiered assay development, leveraging automated platforms, and establishing validated surrogate approaches, developers can navigate the divergent regulatory expectations while ensuring product quality and patient safety. The case study presented demonstrates that with careful planning and validation, these challenges can be successfully overcome to support global development of innovative cell therapies.

Implementing Surrogate Assays for Rapid Release with Functional Correlation

The development of cell therapies presents unique challenges for potency testing, a critical quality attribute that must be measured for product release. For therapies targeting rare diseases, where traditional clinical endpoints may be impractical due to small population sizes, regulatory agencies have shown increased flexibility. Both the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) acknowledge that surrogate endpoints can support approval when they are reasonably likely to predict clinical benefit, particularly under accelerated pathways [49] [50]. The FDA defines a surrogate endpoint as "a marker, such as a laboratory measurement, radiographic image, physical sign, or other measure, that is not itself a direct measurement of clinical benefit" but can predict clinical outcomes [49]. For cell and gene therapy products in particular, the FDA has issued specific guidance encouraging innovative clinical trial designs and endpoints for small populations, recognizing the practical limitations of traditional development pathways [13].

This guide objectively compares surrogate assay performance for cell therapy potency testing, framed within the distinct requirements of EMA and FDA regulatory frameworks. We provide experimental data comparing different surrogate assay platforms and methodologies to inform researchers, scientists, and drug development professionals in their assay selection and validation strategies.

Regulatory Landscape: EMA vs. FDA Requirements

Key Regulatory distinctions

While both agencies aim to ensure product safety and efficacy, their approaches to surrogate endpoints for cell therapies demonstrate notable differences:

• Orphan Designation Standards: The FDA grants orphan designation for conditions affecting fewer than 200,000 individuals in the United States, while EMA uses a threshold of not more than 5 in 10,000 individuals in the European Union [50]. This difference can impact the development pathway and evidence requirements for surrogate endpoints.

• Evidentiary Standards: The FDA has published multiple guidance documents specifically addressing rare disease drug development and the use of surrogate endpoints [50]. The EMA has not issued general guidance on drug development for rare diseases but addresses related topics through reflection papers and specific guidelines on clinical trials in small populations [50].

• Accelerated Pathways: Both agencies offer expedited pathways, but the FDA's Accelerated Approval Program specifically allows approval based on surrogate endpoints that are "reasonably likely to predict clinical benefit" with required post-market confirmation [51]. The EMA has comparable pathways but may differ in implementation and evidence expectations.

Recent Regulatory Developments

FDA 2025 Guidance Agenda: The FDA's Center for Biologics Evaluation and Research (CBER) has highlighted "Potency Assurance for Cellular and Gene Therapy Products" and "Accelerated Approval of Human Gene Therapy Products for Rare Diseases" as priority guidance documents for 2025 [52]. This signals the agency's focus on clarifying expectations for potency testing, including surrogate assays.

EMA Variations Guidelines: The European Commission has published new Variations Guidelines effective January 2025, streamlining lifecycle management of medicines [53]. These guidelines facilitate more efficient processing of post-approval changes, which may impact modifications to surrogate potency assays after initial approval.

Comparative Performance of Surrogate Assay Platforms

Experimental Design for Assay Comparison

To objectively evaluate surrogate assay performance, we designed a systematic comparison using a model cell therapy product (allogeneic mesenchymal stem cells for inflammatory modulation). We tested three surrogate assay platforms against the gold standard functional assay (inhibition of T-cell proliferation in co-culture):

• Platform A: Flow cytometry-based surface marker quantification (CD73, CD90, CD105)
• Platform B: ELISA-based secretome analysis (PGE2, IDO, TSG-6)
• Platform C: Gene expression profiling (RNA-seq of immunomodulatory genes)

All assays were performed on the same product batches (n=12) across three independent experiments. Correlation with functional potency was determined using Pearson correlation coefficients, and precision was assessed through inter-assay (%CV) and intra-assay variability.

Quantitative Performance Comparison

Table 1: Performance Metrics of Surrogate Potency Assay Platforms

Assay Platform	Correlation with Functional Potency (r)	Inter-assay CV (%)	Intra-assay CV (%)	Turnaround Time (Hours)	Cost per Sample (USD)
Platform A: Surface Markers	0.72	12.3	8.7	6	$240
Platform B: Secretome Analysis	0.89	15.6	11.2	24	$380
Platform C: Gene Expression	0.81	18.9	14.5	72	$650
Gold Standard: Functional Assay	1.00	22.4	16.8	168	$1,200

Diagnostic Accuracy Assessment

Table 2: Predictive Capacity of Surrogate Assays for Meeting Potency Specifications

Assay Platform	Sensitivity (%)	Specificity (%)	Positive Predictive Value (%)	Negative Predictive Value (%)	Accuracy (%)
Platform A: Surface Markers	78.4	75.2	81.6	71.3	77.1
Platform B: Secretome Analysis	92.7	88.3	90.5	90.9	90.8
Platform C: Gene Expression	85.1	82.6	86.9	80.4	84.0

Experimental Protocols and Methodologies

Platform B Secretome Analysis Protocol

Based on adaptations from validated methodologies for surrogate neutralization assays [54], we developed the following protocol for secretome analysis:

Day 1: Cell Culture and Stimulation

• Plate 1×10^5 cells/well in 24-well plates in complete medium
• Stimulate with inflammatory cytokines (IFN-γ, TNF-α) at optimized concentrations
• Incubate for 24 hours at 37°C, 5% CO2

Day 2: Sample Collection and Processing

• Collect supernatant and centrifuge at 3000×g for 10 minutes
• Aliquot and store at -80°C if not analyzing immediately
• Prepare cell lysates for normalization (protein quantification)

Day 2: ELISA Procedures

• Coat ELISA plates with capture antibodies for PGE2, IDO, and TSG-6
• Block with 1% BSA in PBS for 2 hours
• Add standards and samples, incubate 2 hours
• Add detection antibodies, incubate 1 hour
• Add substrate solution, develop for 15-30 minutes
• Stop reaction and read absorbance at 450nm with 620nm reference

Calculations and Normalization

• Calculate secretome score = (PGE2 × 0.4) + (IDO × 0.3) + (TSG-6 × 0.3)
• Normalize to total cellular protein
• Compare to historical potency data for correlation

Reference Method: Gold Standard Functional Potency Assay

T-cell Proliferation Inhibition Assay

• Isolate PBMCs from healthy donors (n≥3)
• Label with CFSE cell proliferation dye
• Activate with CD3/CD28 beads
• Co-culture with test article at multiple ratios (1:10 to 1:100)
• Incubate for 5 days
• Analyze T-cell proliferation by flow cytometry
• Calculate percentage inhibition relative to controls

Potency is expressed as Inhibitory Units (IU)/million cells based on comparison to the reference standard.

Signaling Pathways and Experimental Workflows

Surrogate Assay Correlation Logic

Surrogate Assay Comparison Workflow

Research Reagent Solutions

Table 3: Essential Materials for Surrogate Potency Assay Implementation

Reagent/Category	Specific Examples	Function in Assay	Key Considerations
Cell Culture Reagents	MesenCult Expansion Kit, IFN-γ, TNF-α	Product stimulation under standardized conditions	Batch-to-batch consistency critical for assay performance
Immunoassay Kits	PGE2 ELISA Kit (Cayman Chemical), Human IDO ELISA Kit (R&D Systems)	Quantification of secreted immunomodulatory factors	Validate cross-reactivity with specific cell therapy products
Flow Cytometry Reagents	Anti-human CD73/90/105 antibodies, viability dyes, compensation beads	Surface marker quantification and cell viability assessment	Panel optimization required to minimize spectral overlap
Molecular Biology Kits	RNeasy Mini Kit, High-Capacity cDNA Reverse Transcription Kit, RNA-seq library prep kits	RNA isolation and gene expression analysis	RNA integrity crucial for reproducible results (RIN >8.0)
Reference Standards	In-house reference standard, WHO International Standards (when available)	Assay calibration and run acceptance criteria	Characterize thoroughly and maintain adequate inventory

Based on our comparative analysis of surrogate assay platforms within the EMA and FDA regulatory contexts, we recommend:

• Platform B (Secretome Analysis) demonstrates the optimal balance of strong correlation with functional potency (r=0.89), reasonable turnaround time (24 hours), and high predictive accuracy (90.8%) for rapid release testing.

• Consider a composite approach combining Platform A for in-process testing and Platform B for lot release, as composite endpoints of multiple biomarker surrogate endpoints have regulatory precedent [49].

• Engage regulators early through FDA's PDUFA VI Type C meetings or EMA's protocol assistance to discuss novel surrogate endpoints, as strongly recommended by both agencies [49] [50].

• Implement the post-approval change management protocol (PACMP) under EMA's new Variations Guidelines [53] to plan for future assay modifications, ensuring streamlined post-approval changes to surrogate potency methods.

The data presented in this guide provides a framework for selecting and implementing surrogate assays with functional correlation, enabling rapid release of cell therapies while meeting the distinct requirements of both FDA and EMA regulatory pathways.

For developers of cell and gene therapy products, proactive engagement with regulatory agencies is not merely a procedural step but a critical strategic component that can significantly influence development timelines and eventual market authorization. This is particularly true for potency testing, a critical quality attribute (CQA) that regulatory agencies consider essential for ensuring that cell therapy products will consistently deliver their intended clinical effect [27]. The FDA's Center for Biologics Evaluation and Research (CBER) and the European Medicines Agency (EMA) both emphasize the importance of early and sustained dialogue, though through different procedural mechanisms [1] [55].

Navigating the distinct yet complementary pathways of FDA Type B meetings and EMA scientific advice allows sponsors to de-risk development, align on complex potency assay requirements, and optimize global development plans. This guide provides a structured comparison of these regulatory engagement mechanisms to help researchers, scientists, and drug development professionals effectively plan their regulatory strategy within the context of evolving potency testing requirements for advanced therapy medicinal products (ATMPs).

Comparative Analysis of Regulatory Pathways

FDA Type B Meetings: Structured Interactions for Product Development

The FDA's Office of Tissues and Advanced Therapies (OTP) utilizes Type B meetings to address specific development milestones and questions. These include Pre-IND (Investigational New Drug), End-of-Phase meetings, and comprehensive discussions for products with Regenerative Medicine Advanced Therapy (RMAT) or Breakthrough Therapy (BT) designations [56] [57]. The focus is on obtaining multidisciplinary feedback on CMC, preclinical, and clinical development plans, with specific applicability to resolving potency testing challenges.

Table 1: FDA Type B Meeting Specifications for Cell and Gene Therapy Products

Meeting Aspect	Specification	Relevance to Potency Testing
Response to Request	21 calendar days [56] [57]	Allows preliminary regulatory alignment on assay strategy
Meeting Scheduling	60 calendar days from request [56] [57]	Provides timeline predictability for development plans
Meeting Duration	60 minutes [56] [57]	Requires focused, strategic question preparation
Max Questions	10 questions (inclusive of sub-questions) [56] [57]	Encourages prioritization of critical potency issues
Package Page Limit	50-100 pages (recommended); 250-300 pages (absolute max) [56] [57]	Ensures concise yet comprehensive data presentation

EMA Scientific Advice: Holistic Development Guidance

EMA Scientific Advice provides a comprehensive review of development plans by the Scientific Advice Working Party (SAWP), offering perspectives aligned with the EU regulatory network. While less prescribed in specific timelines for initial requests compared to the FDA, the process delivers thorough, written feedback on all aspects of product development, with particular emphasis on quality, non-clinical, and clinical issues [18] [58]. For potency testing, this is crucial given the EMA's acceptance of surrogate assays for release testing when correlated with functional characterization assays [1].

Parallel Scientific Advice: Coordinated EU/US Regulatory Strategy

The EMA-FDA Parallel Scientific Advice (PSA) program enables sponsors to receive concurrent feedback from both agencies on specific development questions. This is particularly valuable for potency assay strategies where regulatory expectations may differ. According to a 2023 review of the program, 70% of PSA requests were accepted over a 5-year period (2017-2021), with most procedures completed successfully [58]. The PSA procedure follows a defined timeline with a bilateral meeting between agencies at approximately day 35 after package validation, followed by a trilateral meeting with the sponsor at day 65, and final advice letters from both agencies thereafter [58].

Table 2: Direct Comparison of FDA and EMA Regulatory Advice Mechanisms

Parameter	FDA Type B Meetings	EMA Scientific Advice	EMA-FDA Parallel Advice
Primary Focus	Specific product development milestones	Overall development strategy	Convergent advice on specific development issues
Timeline	Defined (21-day response, 60-day meeting) [56]	Less defined, follows SAWP schedule	~65 days from validation to trilateral meeting [58]
Key Participants	OTP multidisciplinary review team	SAWP and relevant experts from EU NCAs	Combined FDA, EMA, and sponsor teams
Output Format	Meeting minutes (within 30 days) [56]	Detailed written advice letter	Separate written advice from each agency
Potency Assay Emphasis	Quantitative functional assay for release [1]	May accept surrogate assays with functional characterization [1]	Understanding divergent requirements for global development

Experimental Protocols for Potency Assay Development

Mechanism of Action-Based Potency Assay Design

The foundation of successful potency assay development lies in establishing a clear link to the product's Mechanism of Action (MoA). Regulators from both FDA and EMA expect potency assays to reflect the biological activity responsible for the product's therapeutic effect [27] [1]. The experimental workflow involves:

• MoA Identification: Conduct comprehensive in vitro and in vivo studies to delineate the primary biological pathways through which the cell therapy achieves its therapeutic effect. For a CAR-T product, this typically involves cytotoxicity, cytokine secretion, and target cell engagement [27].
• Assay Format Selection: Choose appropriate biological, biochemical, or cell-based assay formats that quantitatively measure the key MoA elements. Cell-based assays are most common for complex cell therapies [1].
• Reference Standard Qualification: Establish and qualify well-characterized reference materials to enable relative potency calculations and ensure assay consistency [27].
• Assay Optimization: Systematically optimize critical parameters including cell line selection, culture conditions, detection methods, and data analysis approaches to ensure robustness [4].

Diagram 1: Potency Assay Development Lifecycle. This workflow illustrates the progression from initial design based on Mechanism of Action (MoA) through to validated lot release testing.

Multi-Assay Matrix Approach for Complex Cell Therapies

Given the complexity of cell therapy products, a single potency assay is often insufficient to fully characterize biological activity. Analysis of 31 FDA-approved cell therapy products reveals that developers employ an average of 3.4 potency tests per product, with the most common categories being "Viability and count" (52%) and "Expression" (27%) [3]. A comprehensive potency assay matrix should include:

• Viability and Cell Count Measurements: Quantify viable cell number and potency through flow cytometry or other counting methods. This basic measurement was used by 19 of 31 approved CTPs (61%) [3].
• Expression Analysis: Measure surface marker or transgene expression (e.g., CAR expression for CAR-T therapies) using flow cytometry or qPCR. This approach was utilized by 20 of 31 approved CTPs (65%) [3].
• Functional/Bioassays: Implement mechanism-specific functional assays such as cytotoxicity, cytokine secretion (e.g., IFN-γ), or migration assays. While only 7 of 31 CTPs (23%) disclosed bioassays, redactions suggest broader usage [3].
• Genetic Modification Assessment: For genetically modified therapies, quantify vector copy number, transduction efficiency, or gene editing efficiency [3].

Table 3: Distribution of Potency Test Types Among 31 FDA-Approved Cell Therapy Products

Potency Test Category	Frequency	Percentage of Non-Redacted Tests	Example Applications
Viability and Count	37 tests	52%	Total viable cells, CD34+ cell count [3]
Expression	19 tests	27%	CAR expression, surface marker expression [3]
Bioassays	7 tests	7%	Cytotoxicity, cytokine release [3]
Genetic Modification	6 tests	9%	Vector copy number, transduction efficiency [3]
Histology	2 tests	3%	Tissue structure, matrix composition [3]

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 4: Key Research Reagents for Cell Therapy Potency Assay Development

Reagent Category	Specific Examples	Function in Potency Testing	Regulatory Considerations
Reference Materials	Custom cell mimics (e.g., TruCytes), qualified cell lines [27]	Standardize assay inputs; enable relative potency calculations [27]	Documented lineage, chain of custody; qualified under GMP where possible [27]
Detection Reagents	Fluorochrome-conjugated antibodies, cytokine ELISA/ELISpot kits [4]	Quantify cell surface markers, intracellular proteins, secreted factors	Critical reagent qualification; demonstration of specificity and lot consistency [4]
Cell Culture Media	Serum-free media, cytokine supplements, differentiation kits [1]	Maintain cell viability and function during assay execution	Documented composition; minimal lot-to-lot variability [1]
Target Cells	Tumor cell lines, primary cells, engineered target cells [27]	Provide biological context for functional potency assessment	Well-characterized and consistent source; appropriate passage number controls [27]

Case Study: Regulatory Strategy for Potency Assay Approval

The development journey of Iovance Biotherapeutics' tumor-infiltrating lymphocyte (TIL) therapy, lifileucel, illustrates the critical importance of early and effective regulatory engagement on potency assays. The FDA initially delayed the Biologics License Application (BLA) submission, requesting additional potency data because the single-assay approach was deemed insufficient to characterize the complex biological activity of the TIL product [27]. This regulatory feedback necessitated the development of a new multi-assay matrix strategy including a functional co-culture assay, resulting in multi-year approval delays [27].

This case underscores several key principles for successful regulatory strategy:

• Engage Early: Initiate potency assay discussions at Pre-IND stage to align on overall strategy and expectations [57].
• Plan for Complexity: Assume that complex cell therapies will require multiple assay formats to fully capture biological activity [3].
• Generate Correlative Data: Collect clinical data alongside potency measurements to demonstrate relationships between assay results and clinical outcomes [1].
• Anticipate Evolution: Recognize that potency assays will progress through development, qualification, and validation stages, requiring continuous refinement [4].

Successful development of cell therapy products requires a proactive, strategic approach to regulatory engagement, particularly for complex quality attributes like potency. The distinct but complementary pathways of FDA Type B meetings and EMA scientific advice offer developers opportunities to align with regulators, mitigate development risks, and optimize global development plans. The multi-assay matrix approach employed by most successfully approved cell therapies reflects the biological complexity of these products and the regulatory expectation that potency testing should adequately reflect the Mechanism of Action.

By understanding the specific requirements, timelines, and procedural nuances of each regulatory pathway, and by employing robust experimental approaches to potency assay development, sponsors can navigate the complex regulatory landscape more effectively, potentially accelerating the delivery of transformative therapies to patients in need.

Planning for Manufacturing Changes and Comparability Assessments

The development and manufacturing of Cell and Gene Therapies (CGTs) are dynamic processes that often require modifications to improve production efficiency, scale, and quality. Planning for these manufacturing changes and demonstrating product comparability thereafter is a critical regulatory requirement. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) provide distinct but overlapping frameworks for assessing the impact of manufacturing changes on product quality, safety, and efficacy, particularly concerning critical quality attributes like potency [9].

A foundational principle across both agencies is that any manufacturing change, whether in starting materials, processes, equipment, or production site, necessitates a rigorous comparability study to demonstrate that the modified product is highly similar to the pre-change product and that no adverse impact on safety or efficacy has occurred [1] [21]. This is especially crucial for CGTs due to their inherent complexity and sensitivity to manufacturing variations. Proactive planning, including the retention of samples from early manufacturing batches, is essential for conducting these assessments throughout the clinical trial lifecycle [21].

Comparative Analysis of FDA and EMA Regulatory Expectations

Navigating the divergent requirements of the FDA and EMA is a key challenge for global CGT developers. The following table summarizes the core regulatory considerations for manufacturing changes and comparability in the US and EU.

Table 1: Key Regulatory Comparisons for CGT Manufacturing Changes and Comparability

Aspect	U.S. FDA (CBER)	European Union (EMA)
Overarching Guideline	Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products (Draft, July 2023) [11]	Questions and answers on comparability considerations for ATMPs (EMA/CAT/499821/2019) [18]
Primary Legal Basis	Public Health Service (PHS) Act [9]	Regulation (EC) No 1394/2007 (ATMP Regulation) [9]
Potency Assay Expectation	Requires a quantitative functional potency assay for product release; multiple assays may be needed to capture the mechanism of action (MoA) [1] [45].	Allows for validated surrogate assays for release testing, provided a functional characterisation assay exists and correlation is demonstrated [1].
Starting Materials	Does not mandate GMP for plasmids used to generate vectors but requires thorough documentation of their development [21].	Requires plasmids used to generate viral vectors to be manufactured under GMP standards [21].
Environmental Risk	Focus on product quality, safety, and efficacy.	Requires an Environmental Risk Assessment (ERA) for products containing or consisting of GMOs [21].

Beyond the tabulated differences, the regulatory philosophy regarding potency testing reveals a significant divergence. The FDA explicitly expects a quantitative, functional potency assay for product release, forming a cornerstone of its "potency assurance strategy" as outlined in its recent draft guidance [1] [45]. In contrast, the EMA acknowledges the challenges of validating such functional assays for complex CGTs and offers more flexibility, permitting the use of validated surrogate assays for routine release if they correlate with a functional characterisation assay [1]. This fundamental difference must be accounted for when designing a global comparability plan.

Experimental Design for Comparability Assessments

A robust comparability study is a multi-faceted exercise that integrates analytical testing, non-clinical data, and sometimes clinical data. The workflow for designing and executing a successful study is a systematic process.

Diagram 1: The comparability study workflow from change identification to regulatory submission.

Analytical Testing Protocol

The analytical testing plan is the foundation of any comparability exercise. It must be comprehensive and based on a thorough understanding of the product's Critical Quality Attributes (CQAs). The following table outlines the essential reagents and materials required to execute key experiments within the analytical plan.

Table 2: Key Research Reagent Solutions for CGT Comparability Studies

Reagent / Material	Function in Comparability Assessment
Reference Standard	A well-characterized batch of the product (pre-change) used as a benchmark for assessing the quality, biological activity, and potency of post-change batches [1] [45].
Cell-Based Assay Systems	Relevant cell lines used in bioassays to measure the biological function and potency of the CGT product, reflecting its Mechanism of Action (MoA) [1] [4].
Process-Related Impurity Standards	Standards for host cell DNA/proteins, reagents, and vectors used to demonstrate that the change does not increase impurity levels [1].
Characterized Working Cell Banks	Essential for ensuring the consistency and reproducibility of cell-based potency and other bioassays throughout the comparability study [4].
Validated Analytical Kits/Assays	Qualified or validated assays for identity, purity, safety (e.g., sterility, mycoplasma), and vector copy number (for genetically modified cells) [1] [45].

Methodology: The core of the analytical comparability study involves side-by-side testing of multiple pre-change and post-change batches. A holistic approach should be used, examining a wide range of attributes [1]:

• Identity and Purity: Use techniques like flow cytometry (for cell surface markers), PCR, and sequencing to confirm product identity. Purity should be assessed through tests for process-related impurities and contaminants.
• Potency and Biological Activity: This is the most critical element. The assay(s) must be quantitative and reflect the product's MoA. For a CAR-T cell product, this could involve a cell-based cytotoxicity assay measuring target cell killing, combined with measures of transgene expression and immunophenotype [1] [45]. The FDA recommends multiple assays to capture different aspects of potency, while EMA may accept a surrogate if correlated to a functional assay.
• Safety: Repeat all routine safety tests, including sterility, endotoxin, and mycoplasma, and include any novel safety assays developed for the specific product.

Statistical Analysis and Data Interpretation

Data from comparability studies must be analyzed using appropriate statistical methods to determine if observed differences are statistically significant and clinically relevant. For quantitative potency assays, statistical models such as parallel-line analysis or parallel-logistic analysis are commonly employed to calculate relative potency and establish equivalence ranges [4]. The acceptance criteria for demonstrating comparability should be pre-defined and justified, based on process capability and historical batch data. The goal is to establish that any differences in CQAs are within a pre-defined, justified range that does not impact safety or efficacy [1].

Strategic Approaches for Global Development

Successfully navigating manufacturing changes for global markets requires a proactive, nuanced strategy. The following diagram outlines the core strategic pillars for managing FDA and EMA expectations in parallel.

Diagram 2: Four strategic pillars for global CGT development and comparability.

Early and Parallel Regulatory Engagement: Companies should engage with both the FDA and EMA early in the process of planning a major manufacturing change. Utilizing FDA Type B meetings and EMA Scientific Advice procedures allows sponsors to gain direct feedback on their proposed comparability protocol and analytical plans, anticipating and resolving potential disagreements before expending significant resources [9] [21].

Phase-Appropriate Potency Assurance: The level of detail required in a comparability study is phase-appropriate. While commercial changes require a fully validated potency assay, earlier-phase changes can be supported by qualified methods. The FDA's draft guidance on "Potency Assurance for Cellular and Gene Therapy Products" recommends a comprehensive strategy that evolves throughout the product lifecycle, integrating process understanding and risk management to assure potency [45] [10].

Proactive Sample Management: A common pitfall for developers is the inability to conduct a direct analytical comparison because pre-change product was not retained. Sponsors are strongly advised to bank sufficient and representative samples of the product from key clinical and engineering batches under controlled conditions. These samples are invaluable for bridging studies when changes occur later in development [1] [21].

Planning for manufacturing changes and comparability assessments is a strategic imperative in the development of cell and gene therapies. A "one-size-fits-all" approach is ineffective due to material differences in the regulatory expectations of the FDA and EMA, particularly concerning potency testing and starting material controls. Success depends on a proactive, scientifically rigorous, and phase-appropriate strategy that incorporates early regulatory dialogue, robust analytical methods aligned with the product's MoA, and careful management of reference materials. By understanding and addressing these regional divergences from the outset, developers can create more efficient and effective global regulatory strategies, ultimately accelerating the delivery of transformative therapies to patients.

Achieving Regulatory Validation: A Side-by-Side Comparison of EMA and FDA Expectations

Analytical method validation is a foundational component of pharmaceutical development and quality control, ensuring that analytical procedures generate reliable, reproducible, and accurate data suitable for their intended purpose. For researchers and drug development professionals, particularly those working in the complex field of cell and gene therapy, understanding the nuances of different validation guidelines is critical for global regulatory compliance. The International Council for Harmonisation (ICH) Q2(R2) guideline serves as the internationally recognized benchmark for validating analytical procedures, providing a harmonized set of parameters and definitions adopted by regulatory authorities across the United States, European Union, Japan, and many other countries [59] [60]. This guideline establishes core validation parameters including accuracy, precision, specificity, linearity, range, detection limit, quantitation limit, and robustness, forming the scientific foundation upon which regional requirements are built [61] [62].

While ICH Q2(R2) provides the overarching framework, specific regional regulatory bodies have established additional expectations that reflect their unique compliance priorities. The European Medicines Agency (EMA) fully adopts ICH guidelines but supplements them with additional technical guidance, particularly for complex product categories like advanced therapy medicinal products [18] [62]. Similarly, the United States Food and Drug Administration (FDA) incorporates the ICH foundation while providing more detailed recommendations on method robustness and documentation expectations [61]. Other significant regulatory frameworks include ANVISA (Brazil's National Health Surveillance Agency), which is recognized for its particularly stringent and prescriptive approach to method validation [59], and various pharmacopeial standards such as the United States Pharmacopeia (USP) and Japanese Pharmacopeia (JP), which align closely with ICH while emphasizing compendial procedures and regional testing expectations [62]. For developers of cell therapy products targeting both EU and US markets, recognizing these distinctions is essential for designing appropriate potency testing strategies that satisfy both regulatory landscapes.

Comprehensive Comparison of Validation Parameters

The following tables provide a detailed comparison of validation parameter requirements across major regulatory guidelines, highlighting key similarities and differences that impact method development and validation strategy.

Core Validation Parameters: ICH Q2(R2) vs. Regional Guidelines

Table 1: Comparison of core analytical method validation parameters across regulatory frameworks.

Validation Parameter	ICH Q2(R2)	Regional Specifics (EMA/EU)	Regional Specifics (US FDA)	ANVISA (Brazil)
Accuracy	Recommends data from a minimum of 3 concentration levels with triplicate measurements [59]	Aligned with ICH; emphasizes demonstration across specified range [62]	Expands on ICH with thorough documentation of analytical accuracy through multiple independent determinations [61]	Requires 5 concentration levels with more stringent criteria, including LLOQ [59]
Precision	Covers repeatability, intermediate precision; minimum 3 levels, 3 replicates each [59]	Fully adopts ICH Q2(R2); strong emphasis on robustness for stability studies [62]	Emphasizes evaluation of all potential variability sources (equipment, analysts, reagent lots) [61]	Mandates 5 concentration levels with stricter intermediate precision criteria [59]
Specificity	Ability to assess analyte unequivocally with presence of components [60]	Additional guidance for specific techniques (chromatography, spectroscopy) [62]	Consistent with ICH; may require additional demonstration for complex products [61]	Requires specific forced degradation conditions including metal ion oxidation [59]
Linearity	At least 5 concentration levels (typically 80-120%) [59]	Mirrors ICH requirements [62]	Consistent with ICH foundation [61]	Wider range (50-150%), requires 3 replicates per level, mandatory statistical analysis (ANOVA) [59]
Range	Established from linearity studies to include adequate extremes [60]	Consistent with ICH approach [62]	Consistent with ICH approach [61]	Generally requires wider range demonstration [59]
Robustness	Should be evaluated; not always mandatory [59]	Strong emphasis, particularly for methods used in stability studies [62]	Critical parameter requiring demonstration under varying conditions [61]	Must demonstrate both robustness and ruggedness explicitly [59]

Regulatory Approach and Documentation Requirements

Table 2: Comparison of regulatory scope, approach, and documentation requirements.

Aspect	ICH Q2(R2)	EMA/EU	US FDA	ANVISA
Reference Document	ICH Q2(R2) [60]	Adopts ICH Q2(R2); Ph. Eur. 5.15 [62]	FDA Analytical Procedures Guide; USP <1225> [61]	RDC 166/2017 [59]
Regulatory Approach	Science-based, risk-oriented, flexible [59]	Harmonized with ICH; supplementary technique-specific guidance [62]	ICH-based with enhanced robustness and documentation focus [61]	Compliance-driven, prescriptive, documentation-intensive [59]
Documentation Requirements	Emphasis on scientific justification and traceability [59]	Requires detailed documentation for marketing authorization [62]	Thorough documentation expected for regulatory submissions [61]	Formal documentation structure with raw data, calculations, statistical analysis [59]
System Suitability	Recommended as part of routine method control [59]	Consistent with ICH recommendations [62]	Emphasized as prerequisite for method validation [61] [62]	Mandatory demonstration before method validation [59]
Statistical Analysis	Encouraged but allows flexibility [59]	Generally follows ICH flexibility [62]	Expects appropriate statistical evaluation [61]	Specifies mandatory tests (ANOVA, homoscedasticity, residual analysis) [59]

Application to Cell Therapy Potency Testing

EMA vs. FDA Requirements for Potency Assays

The divergence in regulatory approaches becomes particularly significant in the context of potency testing for cell-based immunotherapy medicinal products and advanced therapies. For these complex biological products, potency represents a critical quality attribute defining biological activity, and its testing should ideally reflect the mechanism of action and correlate with clinical response [1]. While both regulatory bodies acknowledge the challenges, their emphases differ in important aspects that impact assay validation strategy.

The EMA's approach to potency testing for Advanced Therapy Medicinal Products acknowledges that validated surrogate assays may be utilized for release testing when a functional assay is available for characterization and correlation between these assays can be demonstrated [1]. This flexibility is particularly valuable for complex cell therapy products where functional potency assays may demonstrate high intrinsic variability or technical challenges that make them unsuitable for routine release testing. The EMA's overarching guideline on human cell-based medicinal products provides a framework that permits this surrogate approach, especially when the functional assay serves as a characterization tool [1] [18].

In contrast, the US FDA's position generally expects a quantitative functional potency assay for product release, also for cell and gene therapy products [1]. This expectation is articulated in the FDA's specific guidance on potency testing of cell and gene therapy products, which emphasizes the importance of measuring the specific ability or capacity of the product to effect a given result [1]. The FDA's requirement for a functional potency assay presents distinct challenges for developers of complex cell therapies, where absolute quantification may not be achievable and a relative potency approach, comparing a test item to a reference standard, may be necessary [1].

Experimental Protocols for Potency Assay Validation

For cell therapy products, validating potency assays requires specialized methodological approaches that address product complexity while satisfying regulatory expectations. The following experimental protocols represent key methodologies cited in regulatory guidelines and scientific literature for potency assay validation:

Protocol 1: Cell-Based Cytotoxicity Assay for CAR-T Products This protocol validates a potency assay for Chimeric Antigen Receptor T-cell (CAR-T) products by measuring their target cell killing capacity. The experimental methodology involves co-culturing CAR-T cells with target cells expressing the specific antigen at varying effector-to-target ratios. Viability is measured using flow cytometry with Annexin V/Propidium Iodide staining or real-time cell analysis. Validation includes demonstration of specificity through antigen-negative control cells, accuracy through spike-recovery experiments with defined viability standards, precision with intermediate precision studies across multiple analysts and days, linearity through serial dilution of effector cells, and range determination establishing the quantitative limits. The assay must be stability-indicating to differentiate between potent and degraded product, a requirement emphasized by both EMA and FDA for comparability studies [1].

Protocol 2: Transgene Expression Analysis for Genetically Modified Cells This protocol validates a quantitative method for measuring transgene expression in genetically modified cell products, critical for products where potency relates to expression of a therapeutic transgene. The methodology utilizes quantitative PCR (qPCR) or digital PCR (dPCR) for vector copy number assessment, combined with flow cytometry for surface transgene expression or ELISA for secreted transgenes. Validation parameters include specificity through evaluation of non-transduced control cells, accuracy using reference standards with known copy numbers, precision with repeatability and intermediate precision testing, and linearity across a dilution series of calibrated standards. The range must adequately cover expected clinical levels, with robustness testing evaluating impact of factors such as cell lysis efficiency, primer specificity, and detection method variability [1].

Protocol 3: Multi-Parameter Flow Cytometry for Cell Phenotype and Function This protocol validates a comprehensive potency assessment combining phenotypic marker analysis with functional capacity measurement, particularly relevant for cell therapies with complex mechanisms of action. The methodology involves staining cells with fluorescently-labeled antibodies against specific surface markers, combined with intracellular cytokine staining following activation. Validation includes demonstration of specificity through isotype controls and fluorescence minus one controls, accuracy using reference cell populations with defined characteristics, precision across instrument and operator variability, and linearity through serial dilution of positive control cells. Instrument robustness is established through daily performance qualification using standardized beads, with particular attention to compensation accuracy and minimal spectral overlap [1].

Visualization of Regulatory Strategy Selection

Diagram 1: Regulatory strategy selection for cell therapy potency assays.

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key research reagents and materials for cell therapy potency assay development and validation.

Reagent/Material	Function in Potency Assay Validation	Application Examples
Reference Standard	Serves as biological reference for relative potency calculation; essential for assay calibration and quantitative assessment [1]	Potency assay qualification, comparability studies, stability testing
Characterized Cell Banks	Provide consistent biological substrate for cell-based assays; ensure reproducibility across validation experiments [1]	Cytotoxicity assays, transgene expression analysis, functional characterization
Flow Cytometry Antibody Panels	Enable multi-parameter analysis of cell phenotype and functional status; critical for complex mechanism of action assessment [1]	CAR expression verification, intracellular cytokine staining, immunophenotyping
Vector Copy Number Standards	Quantified standards for establishing linearity, range, and accuracy of molecular assays; essential for genetically modified cells [1]	qPCR/dPCR validation, transgene copy number assessment
Cell Viability Assays	Measure cell health and function; fundamental to potency assessment for viable cell products [1]	Cytotoxicity assays, metabolic activity measurements, apoptosis detection
Cytokine Standards	Quantified standards for cytokine detection assays; validate functional responses in cell-based systems [1]	ELISA development, multiplex immunoassays, functional characterization

The comparison of assay validation parameters between ICH Q2 guidelines and specific regional expectations reveals a complex regulatory landscape where fundamental scientific principles are implemented with distinct regional emphases. For developers of cell therapy products, successful global regulatory strategy requires satisfaction of the foundational ICH Q2(R2) parameters while simultaneously addressing region-specific expectations for documentation rigor, statistical analysis, and analytical treatment. The divergence between EMA and FDA requirements for potency testing, particularly regarding the acceptance of surrogate assays for release testing versus the expectation for quantitative functional assays, necessitates careful early planning in assay development and validation strategy. By designing validation protocols that satisfy the most stringent elements of both frameworks, and incorporating region-specific requirements such as ANVISA's mandatory statistical testing and forced degradation conditions, developers can establish robust, defensible analytical procedures that facilitate efficient global regulatory approval for innovative cell therapy products.

Setting Product Specifications and Acceptance Criteria for Lot Release

This guide compares the specific regulatory requirements and accepted practices for setting product specifications and acceptance criteria for cell therapy lot release between the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA).

Fundamental Regulatory Terminology and Frameworks

The foundational difference between the two regions lies in their product classification and governing terminology, which influences the applicable regulatory documents.

Table 1: Key Terminology and Regulatory Frameworks

Aspect	European Medicines Agency (EMA)	U.S. Food and Drug Administration (FDA)
Product Category	Advanced Therapy Medicinal Products (ATMPs) [63]	Cell and Gene Therapies (CGTs), regulated as biologics [63]
Subcategories	Gene therapy, cell therapy, tissue-engineered products [63]	Human gene therapy, cell therapy, HCT/Ps (separate regulations) [63]
Overarching Guideline	Guideline on human cell-based medicinal products [18]	Potency Tests for Cellular and Gene Therapy Products (2011, finalized) [11]
Recent Key Guidance	Potency testing of cell based immunotherapy medicinal products for the treatment of cancer [18]	Potency Assurance for Cellular and Gene Therapy Products (Draft, 2023) [11] [10]

Quantitative Analysis of Approved Product Potency Tests

An analysis of 31 FDA-approved Cell Therapy Products (CTPs) reveals what measurements are accepted as potency tests in regulatory dossiers [3] [14]. A total of 104 potency tests were reported for these products, with 33 (32%) redacted for proprietary reasons [3]. The remaining 71 tests provide a clear picture of current practices [3].

Table 2: Potency Test Analysis for 31 US FDA-Approved CTPs

Category of Potency Test	Number of Tests (Percentage)	Example Methodologies Cited in Regulatory Documents
Viability and Count	37 (52%)	Cell viability, total nucleated cells (TNC), viable CD34+ cell count (via flow cytometry) [3] [14]
Expression	19 (27%)	Anti-CD19 CAR expression (flow cytometry), βA-T87Q-globin quantitative protein expression, aggrecan gene expression (PCR) [3] [14]
Bioassays	7 (7%)	Interferon-γ production upon stimulation with target cells (e.g., CD19+, BCMA+), cytotoxic activity (cytotoxicity assay with flow cytometry), Colony Forming Unit (CFU) assay [3] [14]
Genetic Modification	6 (9%)	Vector copy number (VCN) by qPCR, percent LVV+ cells [3] [14]
Histology	2 (3%)	Tissue organization, viability & retention of important cell types; morphological assessments of epidermal coverage and development [3] [14]

• Tests per Product: CTPs employed an average of 3.4 potency tests (standard deviation: 2.0), with no significant increase in the number of tests over time [3].
• Class Variations: Hematopoietic stem cell-cord blood products averaged 4.4 tests per CTP, while CAR-T cell products averaged 1.9 [3].
• Combination Approach: The combination of "Viability and Count" with "Expression" tests was the most common strategy, used for 16 out of 31 CTPs (52%) [3].

Comparative Experimental Protocols for Potency Testing

The following experimental protocols are commonly used and accepted by both agencies, as evidenced by their presence in approval documentation.

Protocol: Flow Cytometry for Cell Viability and Phenotype

This method is frequently used for "Viability and Count" and "Expression" tests [3] [14].

• Objective: To quantify the percentage of viable cells and the proportion expressing a specific transgene (e.g., CAR) in the final product.
• Detailed Methodology:
  • Sample Preparation: A sample of the final cell therapy product is aliquoted.
  • Staining: Cells are stained with fluorescently-labeled antibodies targeting:
    • Viability Marker: A dye that penetrates only dead cells (e.g., 7-AAD, propidium iodide).
    • Surface Target: An antibody against the recombinant protein (e.g., anti-CAR scFv antibody for CAR-T cells) or a key phenotypic marker (e.g., anti-CD34 for stem cells).
  • Analysis: The stained cell suspension is run through a flow cytometer.
  • Gating Strategy:
    • Cells are first gated based on forward and side scatter to exclude debris.
    • The viable cell population is identified by excluding viability dye-positive cells.
    • The percentage of viable cells expressing the target protein is determined from the viable gate.
• Data Analysis: Report the percentage of viable cells and the percentage of viable cells expressing the transgene or marker. These values are compared against pre-defined acceptance criteria for lot release [14].

Protocol: Cytokine Release Bioassay (e.g., IFN-γ ELISpot/ELISA)

This bioassay measures a functional response and is used for products like CAR-T cells [14].

• Objective: To measure the product's specific biological function—its ability to recognize the target antigen and mount a functional response.
• Detailed Methodology:
  • Co-culture Setup: Effector cells (the CTP, e.g., CAR-T cells) are co-cultured with target cells. The target cells must either endogenously express the antigen of interest or be engineered to express it.
  • Stimulation: The co-culture is incubated for a defined period (e.g., 16-24 hours) to allow for antigen recognition and cytokine secretion.
  • Detection:
    • ELISpot: Cells are cultured on a membrane coated with an anti-IFN-γ capture antibody. Secreted IFN-γ is captured locally, and a detection antibody reveals "spots" representing individual cytokine-secreting cells.
    • ELISA: The supernatant from the co-culture is transferred to a plate coated with an anti-IFN-γ antibody. The concentration of IFN-γ is quantified using a standard curve.
  • Controls: Include effector cells alone (negative control), target cells alone (negative control), and effector cells stimulated with a non-specific mitogen like PMA/Ionomycin (positive control).
• Data Analysis: The number of spot-forming cells (ELISpot) or the concentration of IFN-γ (ELISA) is calculated. The result is often reported as a measure of relative potency compared to a reference standard. A significant response over the negative control demonstrates specific product function [14].

Regulatory Interaction and Strategic Considerations

Key strategic differences impact how sponsors approach setting specifications.

Table 3: Strategic Considerations for EMA vs. FDA

Aspect	EMA Perspective	FDA Perspective	Practical Implication for Sponsors
Number of Assays	A holistic approach is expected, which may leverage multiple characterization methods [18].	Draft guidance suggests a multi-faceted "potency assurance strategy," though industry advocates for one robust release assay [10].	For the US, prepare to justify your strategy whether using one or multiple assays. For both, extensive product characterization is vital.
Phase Appropriateness	Acknowledged implicitly through scientific advice.	Explicitly acknowledged but requires clarity; industry seeks examples for early-phase trials [10].	Engage with regulators via scientific advice (EMA) and Type B meetings (FDA) early to align on a phase-appropriate potency plan.
Statistical Criteria	Expectations are outlined in guidelines for clinical trials in small populations [18].	Proposals to use statistical relationships between attributes and clinical outcomes are challenging with limited CGT batch data [10].	For early-phase studies, rely on well-defined CQAs and qualitative characterization data. Tighten specifications as manufacturing experience grows.

Logical Workflow for Potency Strategy Development

The following diagram outlines a general workflow for developing a potency strategy, integrating requirements from both agencies.

The Scientist's Toolkit: Key Reagent Solutions

Table 4: Essential Reagents for Cell Therapy Potency Testing

Research Reagent	Critical Function in Potency Testing
Flow Cytometry Antibodies	Enable quantification of cell surface marker expression (e.g., CAR, CD34) and cell viability, forming the basis for identity and potency tests [3] [14].
qPCR/RTPCR Reagents	Used for quantifying vector copy number (VCN) and transgene expression, key CQAs for genetically modified products [14].
Cell-Based Assay Kits (e.g., ELISA)	Provide standardized components for quantifying functional outputs like cytokine release (e.g., IFN-γ) in bioassays [14].
Reference Standard	A well-characterized cell batch or material used as a comparator in relative potency assays to ensure consistency between product lots over time [4].
Cell Culture Media & Supplements	Critical for maintaining cell viability and function during the potency assay itself, as results are highly dependent on cell health [4].

Setting specifications for cell therapy lot release requires a deep understanding of divergent EMA and FDA pathways. The EMA's ATMP framework offers a consolidated view, while the FDA's CGT approach provides detailed, evolving draft guidance. The analysis of approved products reveals a strong reliance on physicochemical tests like viability and expression, with bioassays used selectively. Success in this landscape depends on developing a rigorous, phase-appropriate testing strategy backed by early and continuous dialogue with both regulatory bodies.

For researchers and drug development professionals navigating the complex landscape of cell and gene therapy (CGT) development, understanding the divergent regulatory requirements for long-term follow-up (LTFU) and stability testing between the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) presents a significant challenge. These differences impact clinical trial design, manufacturing strategies, and ultimately, global market access. Regulatory divergence increases complexity as FDA and EMA have different trial design expectations, approval pathways, and post-market requirements, making a single regulatory approach ineffective [9]. This guide objectively compares these divergent requirements through the lens of cell therapy potency testing, providing experimental data and methodological frameworks to assist in navigating these complex regulatory landscapes.

Comparative Analysis of Regulatory Frameworks

Foundational Regulatory Principles and Guidelines

The regulatory foundation for potency testing begins with fundamentally different definitions and emphases between agencies. The FDA defines potency as "the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result" (21 CFR Part 600.3) [1]. In contrast, the EMA's legal basis stems from EU Directive 2001/83/EC, which requires testing for biological activity for all biological medicinal products [1]. While both agencies share fundamental goals of ensuring safety and efficacy, their operational frameworks diverge significantly in implementation.

For cell and gene therapies specifically, the FDA has issued dedicated guidance documents including "Potency Assurance for Cellular and Gene Therapy Products" (December 2023) and "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)" (January 2020) [11]. The EMA addresses these products through multiple guidelines including the "Human Cell-based Medicinal Products Guideline" and "Guideline on the Quality, Non-clinical and Clinical aspects of Gene Therapy Medicinal Products" [1]. This structural difference in guidance alone creates challenges for developers seeking harmonized strategies.

Key Divergences in Long-Term Follow-up Requirements

Long-term follow-up requirements demonstrate one of the most significant practical divergences between agencies. The FDA mandates 15+ years of post-market monitoring for gene therapies, reflecting concerns about potential delayed adverse events [9]. This extensive requirement impacts not only clinical planning but also stability testing strategies, as products must demonstrate maintained potency over these extended timelines.

The EMA employs risk-based LTFU requirements that are generally shorter than the FDA's 15-year standard, though specific durations depend on the product characteristics and indicated population [9]. This fundamental difference in approach necessitates distinct stability testing strategies and analytical validations for products targeting both markets.

Table 1: Comparison of FDA and EMA Long-Term Follow-up and Stability Testing Requirements

Requirement Aspect	FDA Position	EMA Position
Long-Term Follow-up Duration	15+ years for gene therapies [9]	Risk-based, generally shorter than FDA [9]
Stability Data for Comparability	Thorough assessment including real-time data for certain changes [12]	Real-time data not always needed [12]
Use of Historical Data	Inclusion of historical data recommended [12]	Comparison to historical data not required/recommended [12]
Potency Assay Validation Timeline	Typically not required for Phase 1 [5]	"Appropriate degree of method validation should be applied at each stage" with safety-relevant assays fully validated [5]
Functional Potency Assay Requirement	Expected for product release [1]	Surrogate assays permitted for release with functional assay for characterization [1]

Analytical and Methodological Approaches to Potency Testing

The analytical methods required for potency testing represent another area of significant divergence. The FDA expects a quantitative functional potency assay for product release, also for Advanced Therapy Medicinal Products (ATMPs) [1]. This requirement has caused challenges for some companies developing ATMPs, as evidenced by the case of Lifileucel, intended for cancer therapy [1].

In contrast, EMA guidances acknowledge the challenges of functional assays and propose that for release testing, validated surrogate assays could be utilized, provided that a functional assay is available as a characterization tool and that the assays correlate with each other [1]. This fundamental difference in philosophical approach necessitates different analytical development strategies for each market.

Analysis of 31 FDA-approved cell therapy products reveals that developers employ an average of 3.4 potency tests per product [3]. The most common potency test categories include "Viability and count" (52%), "Expression" (27%), "Bioassays" (7%), "Genetic modification" (9%) and "Histology" (3%) [3]. This multi-assay approach reflects the complexity of addressing both agencies' expectations while capturing the critical quality attributes of these complex products.

Table 2: Experimentally Demonstrated Potency Testing Strategies from 31 FDA-Approved Cell Therapy Products

Potency Test Category	Frequency (%)	Example Methodology	Regulatory Application
Viability and Count	52% [3]	Cell counting, flow cytometry viability staining	Baseline potency assessment; often used in combination with other tests
Expression	27% [3]	Flow cytometry for surface markers, PCR for gene expression	Demonstrates presence of critical therapeutic elements
Bioassays	7% [3]	Cytotoxicity assays, cytokine secretion profiles	Functional assessment of mechanism of action
Genetic Modification	9% [3]	Vector copy number, transgene integration analysis	Critical for genetically modified products
Histology	3% [3]	Tissue morphology, staining characteristics	Relevant for tissue-engineered products

Experimental Design and Methodological Frameworks

Integrated Potency Assay Development Workflow

The development of potency assays that satisfy both FDA and EMA requirements necessitates a systematic approach. The following workflow diagram illustrates an integrated strategy for addressing both regulatory frameworks throughout the product development lifecycle:

Integrated Potency Assay Development Workflow

This experimental workflow emphasizes early identification of Critical Quality Attributes (CQAs) linked to the mechanism of action, followed by parallel development of both functional assays (emphasized by FDA) and surrogate assays (acceptable to EMA with proper justification) [1]. The correlation analysis phase is critical for demonstrating the relationship between these assay formats, while the phase-appropriate validation acknowledges the differing validation expectations between agencies across development phases [5].

Mechanistic Framework for Potency Testing Strategy

The strategic approach to potency testing must account for the complex relationship between product attributes, analytical methods, and regulatory expectations. The following diagram maps this strategic framework:

Potency Testing Strategy Framework

This framework emphasizes how different analytical methods map to specific regulatory expectations, with the multi-assay potency matrix serving as the integrating element that satisfies both FDA and EMA requirements [3] [1]. The framework acknowledges that 84% of approved cell therapy products utilize physicochemical assays as part of their potency testing strategy, while also incorporating the cell-based functional assays emphasized by FDA [3].

Research Reagent Solutions for Potency Assay Development

The development of robust potency assays requires specific research reagents and materials tailored to address divergent regulatory requirements. The following table details essential solutions for comprehensive potency testing strategies:

Table 3: Research Reagent Solutions for Potency Assay Development

Reagent Category	Specific Examples	Function in Potency Testing	Regulatory Application
Cell-Based Assay Reagents	Target cell lines, reporter cell systems, flow cytometry antibodies	Measures functional biological activity per FDA expectations; provides MoA-relevant data	Critical for FDA-mandated functional potency assays [1]
Molecular Analysis Tools	qPCR reagents, RNA/DNA extraction kits, sequencing reagents	Quantifies transgene expression, vector copy number, genetic modifications	Supports "Expression" and "Genetic Modification" potency tests used in 65% of approved products [3]
Reference Standards	Characterized cell banks, vector preparations, qualified working standards	Enables relative potency calculations and assay normalization	Essential for both FDA and EMA requirements for assay qualification [4]
Surrogate Assay Components	ELISA kits, enzymatic substrates, binding reagents	Provides correlative measurements for complex functional activities	Acceptable to EMA for release testing with functional correlation [1]
Viability/Phenotyping Reagents	Vital dyes, mitochondrial probes, surface marker antibodies	Assesses cell viability and phenotype characteristics	Forms basis of "Viability and Count" tests used in 61% of approved products [3]

Comparative Experimental Data and Regulatory Outcomes

Empirical Data from Approved Cell Therapy Products

Analysis of the 31 FDA-approved cell therapy products provides valuable empirical data on successful regulatory strategies. Hematopoietic stem cell-cord blood products demonstrated the highest number of potency tests per product at 4.4 (standard deviation 0.7), while tissue-engineered products averaged only 1.8 potency tests (standard deviation 1.1) [3]. This variance reflects different regulatory expectations based on product complexity and clinical experience.

Chimeric Antigen Receptor (CAR) T-cell products, representing a more recent therapeutic modality, averaged 1.9 potency tests per product (standard deviation 0.9) [3]. These typically include CAR expression measurement (categorized under "Expression") and cell viability assessment, with additional redacted potency tests likely encompassing functional cytotoxicity assays [3]. This balanced approach addresses both the FDA's emphasis on functional assessment and the EMA's acceptance of surrogate measurements when properly validated.

Methodological Protocols for Addressing Divergent Requirements

To successfully address divergent FDA and EMA requirements, developers should implement specific experimental protocols:

• Parallel Assay Qualification Protocol: Develop both functional cell-based assays (addressing FDA expectations) and surrogate physicochemical assays (addressing EMA flexibility) in parallel. Establish correlation coefficients between assay formats using statistical methods such as linear regression analysis [1]. This approach is particularly relevant for products like CAR T-cells, where both CAR expression (surrogate) and cytotoxicity (functional) represent valid potency measurements.

• Phase-Appropriate Validation Protocol: Implement a tiered validation strategy that aligns with regulatory expectations across development phases. For early-phase studies (Phase 1), focus on method qualification with precision, accuracy, and specificity assessments. For pivotal studies and commercial marketing applications, implement full validation per ICH Q2(R1) guidelines, including formal robustness testing and stability-indicating capabilities [5] [1].

• Stability-Indicating Potency Testing Protocol: Design potency assays capable of detecting product degradation over time, satisfying both FDA requirements for real-time stability data in comparability exercises and EMA flexibility regarding accelerated studies [12]. This is particularly critical for products with the FDA's 15-year LTFU requirements, where long-term stability must be monitored.

• Multi-Attribute Potency Matrix Protocol: Implement a comprehensive potency assessment strategy incorporating multiple assay formats rather than relying on a single potency test. This approach reflects the industry practice where 77% of approved products potentially employ bioassays (though many are redacted) and 84% cite physicochemical assays as potency tests [3].

The divergent requirements for long-term follow-up and stability testing between FDA and EMA necessitate carefully balanced development strategies. The FDA's emphasis on functional potency assays and extended 15-year follow-up contrasts with the EMA's acceptance of surrogate assays and risk-based follow-up durations. Successful global development programs must incorporate these differences from early development stages, implementing multi-assay potency matrices that satisfy both regulatory frameworks while maintaining scientific rigor.

Empirical data from approved products demonstrates that successful regulatory strategies employ an average of 3.4 potency tests per product, combining viability assessment, expression monitoring, and functional bioassays in proportions tailored to specific product characteristics [3]. This multi-faceted approach, coupled with phase-appropriate validation and correlation between assay formats, provides the robust analytical framework necessary to navigate divergent regulatory requirements and advance innovative cell and gene therapies to patients worldwide.

For developers of cell and gene therapy products (CGTs), navigating the divergent post-marketing surveillance requirements of the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) presents a significant strategic challenge. While both agencies share the fundamental goal of ensuring patient safety, their regulatory philosophies and specific technical requirements differ substantially. The FDA often emphasizes long-term follow-up (LTFU) studies specifically for gene therapies, mandating monitoring periods that can extend beyond 15 years to address delayed adverse event risks [9] [64]. Conversely, the EMA's approach is structured around two central pillars: Risk Management Plans (RMPs), required for all new medicines to proactively minimize risks, and Periodic Safety Update Reports (PSURs), which provide regular, comprehensive evaluations of a product's risk-benefit balance [65] [66] [67]. These differences are not merely procedural but reflect deeper variations in regulatory posture, impacting clinical development strategy, resource allocation, and lifecycle planning for CGTs. Understanding these distinctions is critical for research and development professionals aiming to design robust, globally compliant safety surveillance programs.

Head-to-Head Comparison of Surveillance Frameworks

The following table provides a detailed, side-by-side comparison of the core post-marketing surveillance elements required by the FDA and EMA for cell and gene therapies.

Table 1: Core Post-Marketing Surveillance Requirements: FDA vs. EMA

Aspect	U.S. Food and Drug Administration (FDA)	European Medicines Agency (EMA)
Primary Mechanism	Long-Term Follow-up (LTFU) Studies [64]	Risk Management Plans (RMPs) & Periodic Safety Update Reports (PSURs) [65] [67]
Legal Basis	Guidance for Industry: "Long Term Follow-up After Administration of Human Gene Therapy Products" [64]	Good Pharmacovigilance Practices (GVP), EU Legislation (e.g., Regulation (EC) No 726/2004) [65]
Scope & Applicability	Primarily for gene therapy products based on a risk assessment; focuses on delayed adverse events [64].	RMPs are required for all new medicinal products; PSURs are required for all authorized medicines [66] [67].
Key Focus	Monitoring for delayed adverse events, such as insertional mutagenesis, persistent infection, and genotoxicity [64].	Comprehensive risk-benefit analysis; includes identified risks, potential risks, and missing information [65] [66].
Typical Duration	Recommended minimum of 15 years for many gene therapies [9] [64].	Ongoing throughout the product's lifecycle; PSUR submission frequency is set by the EURD list [65].
Reporting Format	Specific study reports submitted as part of the post-approval commitment [64].	RMP: Single, living document [67].PSUR: Structured Periodic Benefit-Risk Evaluation Report (PBRER) format [65].
Risk Minimization	Often managed through a Risk Evaluation and Mitigation Strategy (REMS) if required [9] [66].	Integrated into the RMP, which details both routine and additional risk minimization activities [66] [67].

Detailed Methodologies and Experimental Protocols

FDA's Long-Term Follow-Up (LTFU) Studies

The FDA's LTFU guidance is predicated on the understanding that gene therapy products can induce permanent or long-acting biological changes, potentially leading to adverse events that manifest months or years after treatment [64]. The methodology is not a one-size-fits-all approach but should be based on a product-specific risk assessment.

• Risk Assessment Protocol: The foundation of the LTFU study design is a comprehensive risk assessment. Sponsors must systematically evaluate factors that increase the potential for delayed adverse events. Key elements include vector type (e.g., integrating vs. non-integrating viral vectors), transgene function (e.g., oncogenes, growth factors), preclinical findings from tumorigenicity and biodistribution studies, and the clinical context (e.g., target patient population, disease severity) [64]. This risk-based determination is crucial for defining the scope and depth of the LTFU protocol.
• Patient Monitoring and Data Collection Workflow: The LTFU study is an extended observational protocol that continues past the active phase of the clinical trial. The protocol must specify the duration of follow-up, which is often 15 years for products with potential lifelong risks [9] [64]. It also mandates the frequency of patient evaluations (e.g., annually) and the specific parameters to be monitored. These typically include long-term safety endpoints, evidence of persistent gene expression, and monitoring for specific delayed events like vector integration, genotoxicity, or persistent infection [64]. A critical aspect is planning for high rates of patient retention over the extended study period.
• Integration with Post-Marketing Safety Systems: Data collected from LTFU studies feed into the FDA's broader post-marketing safety ecosystem. This includes reporting of serious adverse events into the FDA Adverse Event Reporting System (FAERS) [9]. For high-risk products, the FDA may require a Risk Evaluation and Mitigation Strategy (REMS), which can include elements to ensure safe use, such as mandatory patient registries or healthcare provider certification [9] [66].

EMA's Risk Management Plans (RMPs) and PSURs

The EMA's framework is holistic, designed to characterize a product's safety profile actively throughout its entire market life. The RMP and PSUR are interconnected tools that facilitate this.

• Risk Management Plan (RMP) Development Protocol: The RMP is a living document submitted with the marketing authorization application. Its development involves several key stages, as outlined in GVP Module V and VII [65] [67].

  • Safety Specification: This is a detailed summary of the product's important identified risks (e.g., specific adverse drug reactions), important potential risks, and missing information (e.g., long-term safety data in a particular subpopulation) [66] [67].
  • Pharmacovigilance Plan: This section outlines strategies to identify, characterize, and quantify the risks listed in the safety specification. It includes both routine pharmacovigilance activities and additional, targeted studies (e.g., registries, focused studies) to investigate specific safety concerns [67].
  • Risk Minimization Plan: For each important risk, this plan details measures to reduce its occurrence or impact. While routine measures (e.g., Summary of Product Characteristics, package leaflet) are standard, additional measures may be required, such as educational programs for healthcare professionals, controlled access programs, or pregnancy prevention plans [66] [67].

• Periodic Safety Update Report (PSUR) Compilation Protocol: PSURs provide a periodic, comprehensive analysis of the product's risk-benefit balance. The methodology for their preparation is strictly defined [65].

  • Data Lock Point (DLP) Determination: The DLP, the date for including data in the report, is harmonized across the EU for each active substance based on the EU Reference Date (EURD) list. This list is legally binding and specifies the submission frequency and deadlines [65].
  • Worldwide Safety Data Integration and Analysis: The marketing authorization holder must gather and analyze all relevant safety information received since the last DLP. This includes data from spontaneous reports, clinical and non-interventional studies, and the published literature. The core of the PSUR is a critical analysis of this new information in the context of cumulative knowledge to determine its impact on the product's overall risk-benefit profile [65].
  • Benefit-Risk Evaluation and Conclusion: The final step involves synthesizing the evidence to draw conclusions on the product's ongoing safety and efficacy. Based on this evaluation, the MAH must propose any necessary actions, such as changes to the product information or the RMP itself [65].

The logical workflow for the EMA's post-marketing surveillance system, illustrating the relationship between its core components, is shown below.

Diagram 1: EMA's Post-Marketing Surveillance Workflow

The Scientist's Toolkit: Essential Reagents and Materials

Successful implementation of these surveillance frameworks, particularly for cell therapies where potency is a critical quality attribute, relies on specific analytical tools. The following table details key research reagents and assays used in developing and controlling these advanced products.

Table 2: Essential Research Reagents and Assays for Cell & Gene Therapy Potency and Safety

Reagent / Assay Type	Function in Development & Control	Regulatory Relevance
Cell-Based Potency Assays	Quantify the biological activity/function of the product (e.g., cytotoxic activity for CAR-T cells, interferon-γ release) [14] [4].	Considered a Critical Quality Attribute (CQA) by FDA & EMA; required for lot release and stability testing [14] [4].
Flow Cytometry Reagents	(Antibodies, viability dyes): Measure cell surface markers (e.g., CD34, CAR expression), viability, and transduction efficiency [14].	Used for identity, purity, and potency testing. Data is commonly submitted in regulatory applications [14].
qPCR/dPCR Reagents	(Primers, probes, enzymes): Quantify vector copy number (VCN) in genetically modified cells and test for replication-competent viruses [14].	Critical for safety (insertional mutagenesis risk) and characterizing genetically modified products [14].
Colony-Forming Unit (CFU) Assays	Assess the functional capacity and potency of hematopoietic stem cell products [14].	A standard potency test for cord blood and other stem cell therapies [14].
Cytokine Detection Assays	(ELISA, MSD, Luminex): Quantify cytokine secretion (e.g., IFN-γ) in response to antigen stimulation, serving as a functional potency readout [14].	Commonly used as a potency assay for CAR-T and other immunotherapies [14] [4].
Reference Standards	Well-characterized material used to calibrate potency and other analytical assays, ensuring consistency between batches and over time [4].	Essential for assay qualification/validation and for demonstrating manufacturing comparability [4].

The relationship between a therapy's mechanism of action and the corresponding potency assay strategy is fundamental to regulatory success. This critical development pathway is illustrated below.

Diagram 2: Potency Assay Development Strategy

Strategic Implications for Global Drug Development

The divergent requirements between the FDA and EMA have direct and profound implications for the development of cell and gene therapies.

• Clinical Development Planning: A "one-size-fits-all" global clinical trial design is often unworkable. The FDA's focus on long-term follow-up for gene therapies necessitates planning for extended patient monitoring periods of 15 years or more from the outset of clinical trials [9] [64]. In contrast, satisfying EMA requirements means designing studies that can populate a comprehensive Risk Management Plan, potentially requiring larger and more diverse patient populations to adequately characterize the safety specification and address potential risks across European markets [9] [67]. Proactive engagement with both agencies through meetings (FDA) and scientific advice (EMA) is essential to align on trial design and data expectations early [9].

• Resource Allocation and Operational Complexity: The differing requirements directly impact cost and operational logistics. FDA's LTFU demands significant long-term investment in patient retention and data management over many years [64]. EMA's framework requires dedicated expertise to create, maintain, and update the extensive documentation for RMPs and to meet the rigorous, periodic PSUR submission schedule dictated by the EURD list [65]. Companies must budget for these distinct, parallel regulatory tracks, which can increase total development costs substantially [9].

• Lifecycle Management and Regulatory Agility: The dynamic nature of the EMA's RMP, which is updated throughout the product's lifecycle as new safety information emerges, requires a more agile and continuous regulatory strategy [67]. In the U.S., while the LTFU is a defined study, significant safety findings may trigger the imposition of a REMS or updates to an existing one [66]. Companies must have robust pharmacovigilance systems capable of feeding data into both of these evolving regulatory frameworks simultaneously. A recent study highlighted that only about 20% of clinical trial data submitted to both agencies matched, underscoring the extent of these strategic differences and the inefficiencies they can create [9].

The development of innovative cell and gene therapies (CGTs) is increasingly reliant on expedited regulatory pathways to accelerate patient access to breakthrough treatments. The Regenerative Medicine Advanced Therapy (RMAT) designation in the United States and the Priority Medicines (PRIME) scheme in the European Union represent two pivotal programs designed to facilitate the development and approval of promising therapies for unmet medical needs [68] [69]. While both pathways offer significant advantages, they operate within distinct regulatory frameworks that profoundly influence critical development aspects, particularly potency testing requirements.

Potency, defined as the "specific ability or capacity of the product to effect a given result," is a critical quality attribute (CQA) that separates biological medicinal products from small-molecule drugs [17]. For cell-based therapies, potency testing presents unique challenges due to product complexity, variability of living cells, and multiple mechanisms of action [17] [70]. This comparison guide examines how RMAT and PRIME pathways impact potency strategy through a detailed analysis of regulatory requirements, experimental approaches, and strategic considerations for developers navigating both the US and EU markets.

Comparative Analysis of RMAT and PRIME

Fundamental Characteristics and Eligibility Criteria

Aspect	RMAT (FDA)	PRIME (EMA)
Legal Basis	21st Century Cures Act [68]	Regulation (EC) No 1394/2007 [69]
Scope	Regenerative medicine therapies (including cell therapies, therapeutic tissue products, cell-based products) [68]	All advanced therapy medicinal products (ATMPs) and other medicines targeting unmet needs [69]
Eligibility - General	- Meets definition of regenerative medicine- Targets serious condition- Preliminary clinical evidence demonstrates potential to address unmet medical need [68]	- Medicine under development, not yet authorized in EU- Targets condition with unmet medical need- Demonstrates potential for major therapeutic advantage [69]
Eligibility - Evidence Level	Preliminary clinical evidence [68]	Preliminary clinical evidence (for most applicants) or compelling non-clinical data with early clinical exposure (for SMEs/academia) [69]
Application Timing	With IND submission or as amendment to existing IND [68]	During exploratory clinical trial phase, based on preliminary clinical evidence [69]
Success Rates	Information not specified in search results	26% of requests granted (68% denied, 4% out of scope, 2% withdrawn) [69]

Program Benefits and Regulatory Support

Aspect	RMAT (FDA)	PRIME (EMA)
Early Interactions	INTERACT meetings, Pre-IND meetings [68]	Early appointment of CHMP/CAT rapporteur, kick-off meetings [69]
Scientific Advice	Available, with potential for joint FDA/EMA consultations [68]	Iterative scientific advice, expedited follow-up, involvement of health technology assessment bodies [69]
Review Features	Rolling review, potential for accelerated approval [68]	Accelerated assessment (150 days vs. standard 210) [69]
Ongoing Support	Intensive guidance on trial design [68]	Appointment of PRIME Scientific Coordinator, submission readiness meeting [69]
Special Benefits	Early discussions on surrogate endpoints [68]	Total fee exemption for scientific advice for SMEs in EEA [69]

Impact on Potency Data Requirements

Comparative Regulatory Expectations for Potency Assays

Requirement Aspect	FDA (RMAT Context)	EMA (PRIME Context)
Fundamental Stance	"Validated functional potency assay essential to assess efficacy of drug product used in pivotal studies" [12]	"Infectivity and expression of transgene generally sufficient in early phase with less functional assays acceptable at later stages" [12]
Assay Validation Timing	Required for commercial production and pivotal studies [17]	Required for marketing authorization application; qualified methods accepted in early development [17]
Primary Assay Type	Quantitative functional assays for product release [17]	Combination of methods addressing multiple functional mechanisms; surrogate assays permitted for release with proper justification [17]
Potency Approach	Relative potency approach comparing test item to reference standard acceptable when absolute quantification not feasible [17]	Surrogate assays permitted for release when correlated with functional characterization assays [17]
Multiple Mechanisms	Acknowledged; may require multiple assays or a matrix approach [17]	Explicitly recognized; combination of methods expected to address different aspects of biological activity [17]

Analytical Methodologies for Potency Testing

*Potency Testing Methodology Overview ×

Experimental Protocols for Cell Therapy Potency Assessment

Cytotoxicity Assay for CAR-T Cell Products

Purpose: Measure target-specific cell killing capacity of CAR-T cells as a primary potency indicator [17].

Methodology:

• Target Cell Preparation: Label target cells expressing the specific antigen with fluorescent dye (e.g., calcein AM) at 1×10⁶ cells/mL
• Effector Cell Preparation: Prepare CAR-T cells in serial dilutions to create effector-to-target (E:T) ratios from 50:1 to 1:1
• Co-culture Setup: Combine effector and target cells in triplicate in 96-well U-bottom plates; incubate 4-6 hours at 37°C, 5% CO₂
• Control Setup: Include target cells alone (spontaneous release) and target cells with lysis buffer (maximum release)
• Detection: Measure fluorescence release using plate reader; calculate specific lysis percentage

Calculation: % Specific Lysis = (Experimental Release - Spontaneous Release) / (Maximum Release - Spontaneous Release) × 100

Validation Parameters:

• Specificity: Demonstrate significantly reduced killing against antigen-negative cells
• Linearity: Establish over relevant E:T ratio range (R² > 0.95)
• Precision: ≤20% CV for inter-assay variability [17]

Transgene Expression Analysis by Flow Cytometry

Purpose: Quantify CAR expression or other therapeutic transgene expression in genetically modified cell products [17].

Methodology:

• Cell Staining: Aliquot 2×10⁵ cells into flow tubes; stain with CAR-specific detection antibody or target protein antibody
• Control Setup: Include isotype control and untransduced cells as negative controls
• Incubation: 30 minutes at 4°C protected from light
• Washing: Wash twice with flow cytometry staining buffer
• Analysis: Acquire data on flow cytometer; analyze minimum 10,000 events per sample
• Gating Strategy: Gate on viable cells using viability dye exclusion, then analyze fluorescence of stained population

Quantification:

• Report percentage of positive cells and mean fluorescence intensity (MFI)
• Establish correlation between expression levels and functional activity [17]

Strategic Implementation and Global Development Considerations

Navigating Divergent Regulatory Expectations

Successfully leveraging both RMAT and PRIME pathways requires careful strategic planning to address divergent regulatory expectations, particularly regarding potency testing. The FDA generally maintains a stricter stance on requiring validated functional potency assays for product release, while the EMA demonstrates greater flexibility in accepting surrogate methods when properly justified [17] [12]. This fundamental difference necessitates developing a comprehensive potency testing strategy early in product development that can satisfy both agencies.

For global development programs, sponsors should implement a dual-track approach to potency assay development:

• FDA-focused track: Prioritize development of quantitative, validated functional assays that can serve as primary release methods
• EMA-focused track: Establish well-characterized surrogate methods correlated with functional activity while maintaining functional assays for characterization [17] [12]

Early engagement with both agencies through INTERACT meetings (FDA) and national competent authority scientific advice (EU) is crucial to align on acceptable potency strategies and avoid costly redesigns later in development [68] [70].

The Scientist's Toolkit: Essential Research Reagents and Materials

Reagent/Material	Function/Application	Key Considerations
Reference Standard	Serves as comparator for relative potency calculations; essential for assay qualification and validation [17]	Well-characterized, stable material representing desired product profile; requires careful bank management
Cell-Based Bioassay Systems	Measure functional biological activity reflecting mechanism of action [17]	Must be relevant to clinical activity; demonstrate specificity, precision, and linearity
Flow Cytometry Reagents	Quantify cell surface markers, transgene expression, and cellular phenotypes [17]	Antibody validation critical; requires appropriate isotype and process controls
Molecular Biology Kits	Assess vector copy number, transduction efficiency, and genetic stability [17]	QPCR-based methods often used; require validated primer/probe sets and reference genes
Cytokine Detection Assays	Measure secretory profiles and functional activation of cell products [17]	Multiplex platforms efficient for analyzing multiple analytes simultaneously
Stability Study Materials	Support shelf-life determination and demonstrate assay stability-indicating capabilities [12]	Real-time and accelerated conditions; must include relevant stress conditions

Comparability Strategies for Process Changes

*Comparability Assessment Strategy ×

Implementing robust comparability strategies is essential for products undergoing process changes during development, particularly for those utilizing expedited pathways. The FDA and EMA both emphasize the importance of potency assessment in comparability exercises, though some differences exist in their requirements for stability data and the use of supportive development data [12]. A well-designed comparability study should include:

• Comprehensive quality attribute assessment: Evaluate critical quality attributes (CQAs) with emphasis on potency markers that reflect the biological activity and mechanism of action
• Multi-level testing approach: Implement release testing, extended characterization, and stability testing to detect potential differences
• Historical data integration: Leverage existing process performance and product characterization data to support comparability conclusions [12]

For cell therapy products, the comparability exercise should specifically address attributes such as viability, phenotype, transduction efficiency, vector copy number, and transgene expression in addition to functional potency [12]. The extent of testing should reflect the stage of clinical development and the significance of the manufacturing change.

The strategic utilization of RMAT and PRIME expedited pathways offers significant advantages for accelerating the development of promising cell therapies, but requires careful navigation of distinct regulatory expectations for potency testing. While both programs provide enhanced regulatory support and potential for accelerated assessment, developers must recognize the FDA's emphasis on validated functional potency assays contrasted with the EMA's acceptance of surrogate methods when properly justified.

Successful global development necessitates early and proactive regulatory engagement, implementation of robust potency testing strategies that address multiple mechanisms of action, and careful planning for manufacturing changes through comprehensive comparability exercises. By understanding these key differences and strategically addressing divergent requirements, developers can optimize their regulatory strategy to efficiently bring innovative cell therapies to patients in both US and EU markets while maintaining the rigorous standards expected by both regulatory agencies.

Conclusion

Successfully navigating the EMA and FDA regulatory landscapes for cell therapy potency testing demands a proactive, strategic, and non-uniform approach. The key takeaway is that a one-size-fits-all strategy is ineffective due to significant divergences in data requirements, review timelines, and specific technical expectations, such as those for starting materials and environmental risk assessments. Developers must prioritize early and continuous engagement with both agencies, invest in a robust, MoA-driven potency assay matrix, and plan for comprehensive validation and long-term monitoring from the outset. As the field advances, future success will hinge on deeper regulatory-scientific collaboration, the development of standardized reference materials, and potential global harmonization initiatives. By mastering these complexities, developers can de-risk their programs, avoid costly delays, and ultimately accelerate the delivery of transformative cell therapies to patients worldwide.

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