Cell Therapy Potency Assays: A Comprehensive Guide from Development to Regulatory Success

Abstract

This article provides a comprehensive guide for researchers, scientists, and drug development professionals on the development, implementation, and validation of potency assays for cell therapies. It covers foundational principles, regulatory requirements, and the critical role of potency as a key quality attribute. Readers will explore diverse methodological approaches, including insights from FDA-approved products, and learn strategies for troubleshooting and optimizing assays for robustness. The guide also details the rigorous validation process required for regulatory compliance and examines emerging trends, such as multi-omics and assay matrices, that are shaping the future of potency testing. By synthesizing current regulatory expectations, real-world case studies, and advanced methodologies, this resource aims to equip developers with the knowledge to accelerate cell therapy progression from bench to bedside.

Understanding Potency Assays: The Cornerstone of Cell Therapy Quality and Regulation

Potency is a critical quality attribute (CQA) that distinguishes biological medicinal products, including cell and gene therapies, from small-molecule drugs. According to regulatory definitions, potency represents "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]. For cell and gene therapy products, potency testing presents unique challenges due to their complex nature, viability requirements, and frequently multifaceted mechanisms of action (MoA) [2] [1]. Unlike chemical drugs with straightforward structures, the biological activity of living therapies must be quantitatively measured through robust assays that ideally reflect the product's clinical response [3] [1]. This comparison guide examines current potency assay technologies, their applications across different advanced therapy medicinal products (ATMPs), and the experimental frameworks used to generate reliable potency data.

Regulatory Framework and Key Definitions

Regulatory agencies require potency testing for all biological medicinal products. The U.S. FDA mandates a quantitative functional potency assay for product release, while the European Medicines Agency (EMA) may accept validated surrogate assays for release under certain conditions, provided a functional assay is available for characterization [1]. Potency assays must be stability-indicating and able to differentiate between intact and degraded product [1]. For commercial production and pivotal clinical trials, validated assays must be in place, though qualified methods are generally accepted during early clinical development [1].

The Mechanism of Action (MoA) Principle

A fundamental principle in potency assay development is the direct linkage to the product's mechanism of action (MoA) [4] [1]. The assay should measure the specific biological activity responsible for the therapeutic effect rather than merely correlative attributes. For example, while cell viability and phenotype are important quality attributes, they are generally insufficient alone to fully address potency for cellular therapies [1].

Relative Potency and Reportable Results

Instead of absolute quantification, most potency assays measure relative potency (RP), which compares the biological activity of a test sample against a well-characterized reference standard (RS) [3]. This approach helps control intra-lab and inter-lab variability. The reportable potency value can be derived from a single valid assay run or averaged across multiple runs to improve accuracy and precision [3].

Comparative Analysis of Potency Assay Technologies

Cytotoxicity Assays for Immune Effector Cells

For immune effector cells such as CAR-T cells, cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells, cytotoxicity represents a primary mechanism of action [5]. The table below compares major assay formats used to measure cell-mediated cytotoxicity.

Table 1: Comparison of Cytotoxicity Assay Methods

Assay Type	Measurement Principle	Key Advantages	Key Limitations	Therapeutic Applications
Chromium-51 Release	Release of radioactive Cr-51 from damaged target cells [6]	Considered gold standard; highly sensitive [6]	Radioactive hazards; short half-life; spontaneous release [6]	CAR-T cells [5], CTLs [5]
Bioluminescence Imaging	ATP-based luciferase activity in viable cells [6]	Non-radioactive; high sensitivity; real-time monitoring [6]	Requires genetically modified target cells; equipment costs [6]	CAR-T cells [6]
Impedance-Based (xCELLigence)	Electrical impedance changes reflecting cell adhesion/viability [6]	Label-free; real-time kinetic monitoring [6]	Specialized equipment; lower throughput [6]	CAR-T cells [6], NK cells [6]
Flow Cytometry-Based	Propidium iodide/annexin V staining of dead/apoptotic cells [6]	Multiplexing capability; distinguishes apoptotic pathways [6]	Complex data analysis; higher technical expertise [6]	γδ T cells [5], CAR-T cells [5]
Enzyme Release (LDH)	Lactate dehydrogenase release from damaged cells [5]	Non-radioactive; colorimetric readout [5]	Background interference from serum [6]	General cytotoxicity screening [5]

Multi-Omics Approaches for CAR T-Cell Characterization

Advanced multi-omics approaches provide comprehensive profiling of cell therapy products beyond traditional functional assays. These methodologies offer deeper insights into the molecular characteristics correlating with clinical responses [4].

Table 2: Multi-Omics Profiling Technologies for CAR T-Cell Potency Assessment

Analytical Domain	Key Measured Parameters	Technology Platforms	Relevance to Potency
Genomics	Vector copy number (VCN), TCR repertoire, vector integration sites [4]	ddPCR, bulk/single-cell TCR sequencing, INSPIIRED pipeline [4]	VCN is mandatory for FDA-approved products; TCR diversity correlates with clinical response [4]
Epigenomics	DNA methylation, histone modifications, chromatin accessibility [4]	Bulk/single-cell ATAC-seq, ChIP-seq [4]	Defines T-cell differentiation states; predictive of persistence [4]
Transcriptomics	Gene expression patterns, transcriptional phenotypes [4]	Bulk/single-cell RNA-seq, microarrays [4]	Identifies exhaustion signatures; stem-like memory programs [4]
Proteomics	Surface marker expression, intracellular signaling proteins [4]	Flow cytometry, mass cytometry, Western blot [4]	Quantifies CAR expression; activation markers (CD107a); cytokines [4] [5]
Metabolomics	Energy metabolism, metabolic intermediates [4]	LC-MS, GC-MS, extracellular flux analysis [4]	Assesses mitochondrial fitness; glycolytic capacity [4]

Cell-Based Potency Assays for Gene Therapy Products

For gene therapy products such as adeno-associated virus (AAV) vectors, potency assays must measure the biological activity of the delivered transgene. The validated potency assay for AAV2-hRPE65v2 (Luxturna) provides an exemplary model [7].

Table 3: AAV2-hRPE65v2 Potency Assay Validation Parameters

Validation Parameter	Acceptance Criteria	Experimental Outcome
System and Sample Suitability	3PL model fit with 9 MOIs; 90% CI for relative potency within 76%-130% [7]	Equivalence tests must pass; failures lead to assay rejection [7]
Specificity	Formulation buffer shows no dose-response; RS vector produces clear dose-dependent response [7]	PLA software estimates zero relative potency for formulation buffer [7]
Dilutional Linearity	R² ≥ 0.85; Slope 0.797-1.049; observed RP aligns with expected values (50%-150%) [7]	Fitted line closely follows line of identity [7]
Intermediate Precision	%GCV <30% for each concentration level [7]	Pooled %GCV for all 20 RP values: 8.2% [7]
Relative Accuracy	Relative bias within ±15% at all tested levels [7]	Point estimates: 50% (+5.9%), 75% (+1.0%), 100% (-0.1%) [7]
Range	50% to 150% of nominal vector concentrations [7]	MOIs from 1×10⁴ to 1.28×10⁶ vg/cell [7]
Robustness	Transduction time (22h ± 4h), sonication pulses (2 ± 1) [7]	Relative potency 94.3%-112%; RSD ≤6.89% [7]

Experimental Protocols for Key Potency Assays

Cytotoxicity Assay Protocol: Flow Cytometry-Based Method

This protocol measures CAR T-cell-mediated cytotoxicity using flow cytometry with annexin V/propidium iodide (PI) staining [6] [5].

Materials and Reagents:

• Target cells (appropriate antigen-positive cell line)
• Effector CAR T-cells
• Annexin V binding buffer
• Fluorescently labeled annexin V
• Propidium iodide (PI)
• Flow cytometer with appropriate laser/filter configuration

Procedure:

• Target Cell Preparation: Harvest and wash target cells, resuspend in assay medium at 1×10⁶ cells/mL.
• Effector Cell Preparation: Wash and count CAR T-cells, serially dilute to achieve desired effector-to-target (E:T) ratios (e.g., 40:1, 20:1, 10:1, 5:1).
• Co-culture Setup: Combine 100μL target cells (1×10⁵ cells) with 100μL effector cells in U-bottom 96-well plates. Include target cells alone (spontaneous death control) and target cells with lysis buffer (maximum death control).
• Incubation: Incubate plates for 4-6 hours at 37°C, 5% COâ‚‚.
• Staining: Transfer cells to FACS tubes, wash with PBS, resuspend in annexin V binding buffer. Add annexin V and PI, incubate 15 minutes in dark.
• Acquisition and Analysis: Analyze samples by flow cytometry within 1 hour. Calculate specific lysis using the formula: (Experimental % death - Spontaneous % death) / (Maximum % death - Spontaneous % death) × 100 [6].

AAV Potency Assay Protocol: Enzymatic Activity Measurement

This protocol details the relative potency assay for AAV2-hRPE65v2, which measures the isomerohydrolase activity of the RPE65 protein [7].

Materials and Reagents:

• HEK293-LRAT cells (genetically modified to express lecithin retinol acyltransferase)
• AAV2-hRPE65v2 reference standard and test articles
• All-trans-retinol (at-ROL)
• Cellular retinaldehyde-binding protein (CRALBP)
• LC-MS/MS system for 11-cis-retinol quantification

Procedure:

• Cell Transduction: Seed HEK293-LRAT cells in appropriate plates. Transduce with AAV2-hRPE65v2 at nine different multiplicities of infection (MOIs) ranging from 1×10⁴ to 1.28×10⁶ vg/cell for both reference standard and test articles.
• Cell Lysate Preparation: After appropriate incubation, harvest cells and prepare lysates. Quantify protein concentration to normalize samples.
• Enzymatic Assay: Incubate cell lysates with non-radioactive at-ROL and CRALBP to enable the conversion to 11-cis-ROL via the isomerohydrolase activity of RPE65.
• Product Quantification: Detect and quantify 11-cis-ROL using LC-MS/MS with appropriate calibration standards.
• Data Analysis: Generate three-parameter logistic (3PL) response curves by plotting the square root transformation of 11-cis-ROL concentrations against log₁₀(MOI). Calculate relative potency using parallel line analysis (PLA) software [7].

Visualizing Potency Testing Workflows

Comprehensive Potency Assessment Strategy for CAR T-Cells

Diagram 1: CAR T-Cell Potency Assessment Strategy. This workflow integrates functional assays with multi-omics characterization to comprehensively evaluate product potency.

Relative Potency Assay Principle

Diagram 2: Relative Potency Assay Workflow. This process compares test articles against reference standards to determine relative biological activity.

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 4: Key Research Reagents for Cell Therapy Potency Assays

Reagent Category	Specific Examples	Function in Potency Testing	Application Notes
Cell Lines	HEK293-LRAT [7], Antigen-positive target cells [6] [5]	Provide cellular context for functional assays; enable transduction efficiency assessment	Select based on relevant antigen expression and susceptibility to effector mechanisms
Cytokines/Growth Factors	IL-2, IL-7, IL-15 [5]	Maintain cell viability and function during assay; simulate in vivo microenvironment	Concentration optimization critical to avoid artificial activation or suppression
Detection Antibodies	Anti-IFN-γ, anti-CD107a, fluorochrome-conjugated secondary antibodies [5]	Enable measurement of activation markers and cytokine secretion	Multiplexing requires careful fluorochrome selection to minimize spectral overlap
Viability/Cytotoxicity Probes	Annexin V, propidium iodide, calcein-AM [6], Cr-51 [6]	Distinguish live/apoptotic/necrotic cells; quantify target cell killing	Consider safety and disposal requirements for radioactive isotopes
Reference Standards	Well-characterized drug lot of known potency [3]	Enable relative potency calculation; control inter-assay variability	Requires careful characterization and long-term stability monitoring
Analytical Standards	11-cis-retinol for LC-MS/MS [7]	Quantify enzymatic reaction products; calibrate instrumentation	Purity and stability critically impact assay accuracy
Povafonidine	Povafonidine, CAS:177843-85-5, MF:C11H13N5, MW:215.25 g/mol	Chemical Reagent	Bench Chemicals
Guanosine-8-d-1	Guanosine-8-d-1 Deuterated Standard|Supplier	Guanosine-8-d-1 is a stable isotope-labeled internal standard for precise bioanalytical research. This product is for Research Use Only. Not for human or veterinary use.	Bench Chemicals

The field of potency testing for cell and gene therapies continues to evolve as products grow more complex. While traditional cytotoxicity assays remain fundamental for immune effector cells, multi-omics approaches provide unprecedented resolution for characterizing product heterogeneity and identifying critical quality attributes [4]. The regulatory emphasis on mechanism of action-based assays drives innovation in assay design, particularly for products with multiple biological functions. Successful potency strategy requires early development of robust, quantitative methods that can be validated for product release and demonstrate correlation with clinical outcomes [1]. As the industry moves toward standardized approaches, the integration of novel analytical technologies with traditional functional assays will ensure comprehensive product characterization while maintaining regulatory compliance.

Potency, defined as the quantitative measure of the biological activity of a drug product, represents a fundamental Critical Quality Attribute (CQA) that fundamentally separates biological medicinal products from those based on small molecules [1]. According to international regulatory guidance, potency must be linked to the relevant biological properties of the product and ideally should correlate with the clinical response [1]. For cell and gene therapy products (CGTs), also called Advanced Therapy Medicinal Products (ATMPs), potency testing presents unique challenges due to their complex nature, which involves nucleic acids, viral vectors, and viable cells as starting materials [1].

The measurement of potency is not merely an academic exercise but a legal requirement for lot release testing of biologics intended for human administration [3]. Regulatory agencies including the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) mandate that potency assays provide a quantitative measure of a drug's intended biological activity based on its mechanism of action (MoA) [3] [4]. This regulatory imperative stems from the critical need to ensure that each manufactured batch possesses the therapeutic activity required for clinical efficacy while maintaining consistency in manufacturing and stability throughout the product's shelf life [1] [8].

Regulatory Expectations and Framework

Global Regulatory Requirements

Regulatory agencies worldwide have established clear legal frameworks requiring potency testing for biological medicinal products. The U.S. Federal Regulation 21 CFR Part 600.3 states that "potency is interpreted to mean 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]. Similarly, the EU Directive 2001/83/EC specifies requirements for biological activity testing in section 3.2.2.1 of Part I of the Annex I [1].

The regulatory expectations for potency tests evolve throughout product development. While qualified methods are generally accepted during early clinical development, validated assays must be in place for commercial production and are recommended before pivotal clinical studies [1]. This phased approach allows developers to establish correlation between potency test results and clinical efficacy, which is particularly important for complex ATMPs that may have multiple mechanisms of action [1].

Differences in FDA and EMA Approaches

While both major regulatory agencies recognize the critical importance of potency testing, some differences exist in their approaches. The US FDA typically expects a quantitative functional potency assay for product release, also for ATMPs [1]. In contrast, EU guidances acknowledge the challenges of functional assays and may allow use of validated surrogate assays for release testing, provided that a functional assay is available as a characterization tool and that correlation between the assays can be demonstrated [1].

Recent analyses of Marketing Authorization Applications (MAAs) in the EU reveal that major issues with potency tests were noted in almost 50% of all ATMP applications, highlighting the significant challenges developers face in meeting regulatory expectations [1]. Both agencies have issued specific guidelines for emerging product categories such as Chimeric Antigen Receptor T-cells (CAR-Ts) and Genome Editing (GE) products, indicating the evolving regulatory landscape for advanced therapies [1].

Analysis of Current Practices in Approved Cell Therapy Products

Potency Test Utilization in FDA-Approved Products

A comprehensive analysis of the 31 U.S. FDA-approved cell therapy products (CTPs) reveals valuable insights into current potency testing practices. Based on regulatory documentation, these products utilize an average of 3.4 potency tests per CTP (standard deviation 2.0), with a total of 104 potency tests reported across all products [8] [9]. Of these, 33 tests (32%) are redacted in public documents, likely for proprietary reasons, leaving 71 non-redacted potency tests available for analysis [8].

The distribution of potency tests across different types of CTPs varies significantly, as shown in Table 1:

Table 1: Number of Potency Tests by CTP Category

CTP Category	Number of Products	Average Number of Potency Tests	Standard Deviation
Hematopoietic Stem Cell-Cord Blood	5	4.4	0.7
CAR T-cell Products	7	1.9	0.9
Tissue Engineered Products	5	1.8	1.1
All CTPs	31	3.4	2.0

[8]

Notably, the number of potency tests per product has not significantly increased over time, despite scientific advances and regulatory emphasis on comprehensive potency testing strategies [8].

Types of Measurements Used in Potency Testing

The 71 non-redacted potency tests from approved CTPs can be categorized into five primary types of measurements, with "Viability and count" and "Expression" assays dominating current practice (Table 2).

Table 2: Distribution of Potency Test Types in FDA-Approved CTPs

Potency Test Category	Number of Tests	Percentage	Description
Viability and count	37	52%	Cell viability, total nucleated cells, viable CD34+ cell count
Expression	19	27%	CAR expression, protein expression, gene expression
Bioassays	7	7%	Cytokine release, cytotoxicity, colony forming units
Genetic modification	6	9%	Vector copy number, percent LVV+ cells
Histology	2	3%	Tissue organization, morphological assessments

[8] [9]

Measurements of gene or protein expression were used by 20 of the 31 CTPs (65%), while 19 CTPs (61%) used measurements of cell viability or cell count as a potency test [8]. "Viability and count" and "Expression" are the two tests most frequently used together for the same product, occurring for 16 CTPs (52%) [8]. Despite regulatory encouragement of functional bioassays, only 7 of 31 CTPs (23%) publicly reported using them, though redacted information suggests this number could be higher [8].

Methodological Approaches to Potency Testing

Relative versus Absolute Potency Assays

The majority of potency assays employed for biological products utilize relative potency (RP) methodology rather than absolute potency measurements [10]. Relative potency assays measure the biological activity of a test sample against a reference standard under the same experimental conditions, significantly reducing variability caused by biological or experimental factors [10].

The fundamental assumption of parallelism must be met for meaningful derivation of %RP, ensuring that the dose-response curves of the reference standard and test samples have similar shapes [3]. This allows the horizontal shift between curves on the dose scale to accurately reflect differences in potency, typically measured at the EC50 (estimated concentration producing 50% of maximum response) [3]. Relative measurement against a reference standard helps control both intra-lab (day-to-day, analyst-to-analyst) and inter-lab assay variability [3].

While absolute potency assays avoid the need for a reference standard, they are more vulnerable to experimental variability and are rarely used for regulatory release testing [10]. Absolute potency may be considered when reference standards deteriorate quickly, but the increased variability often leads to more failed assays and wasted resources [10].

Orthogonal Approach for Comprehensive Characterization

Leading regulatory agencies recommend an orthogonal approach employing multiple independent methods to assess critical quality attributes like potency, identity, and purity [11]. This methodology uses diverse analytical techniques that provide complementary information, allowing for comprehensive product characterization while reducing the risk of false negative or false positive results [11].

For cell-based therapies, orthogonal identity testing combines genotypic, phenotypic, and morphological analyses to unambiguously identify the finished product [11]. This approach is particularly important for autologous products, where confirmation that cells originated from a specific patient is essential, and for allogeneic products, where major histocompatibility complex antigens must be characterized to prevent rejection [11].

Diagram 1: Orthogonal approach for comprehensive cell therapy product characterization

Advanced Methodologies for CAR T-Cell Potency Assessment

CAR T-cell products represent a particularly complex category where potency assay development has evolved significantly. Traditional potency assays for FDA-approved CAR T-cell products primarily measure IFN-γ release in response to target cells, along with cell viability and CAR expression [4]. However, recent advances in multi-omics approaches have revealed a broader spectrum of important CAR T-cell characteristics that correlate with function [4].

Genomic profiling now includes not only vector copy number (VCN) quantification, which is a mandatory component of lot-release testing, but also vector integration site analysis and T-cell receptor (TCR) sequencing [4]. The recent FDA investigation into cases of secondary malignancies in patients who received CAR T-cell therapy has heightened attention to insertional mutagenesis risks, making integration site analysis increasingly important for both safety and efficacy assessment [4].

Epigenomic profiling through DNA methylation analysis and chromatin accessibility assays provides insights into CAR T-cell differentiation states, which have been shown to critically influence therapeutic efficacy [4]. Single-cell technologies enable simultaneous analysis of transcriptional phenotypes and TCR clonotypes, allowing researchers to identify functional T-cell states that correlate with positive clinical outcomes [4].

Diagram 2: Multi-omics approaches for comprehensive CAR T-cell potency assessment

Essential Research Reagents and Methodologies

The development and implementation of robust potency assays requires specific research reagents and methodological approaches tailored to product characteristics and mechanism of action. Table 3 details key solutions essential for potency assay development and execution.

Table 3: Essential Research Reagent Solutions for Potency Assay Development

Reagent/Method	Function in Potency Assessment	Example Applications
Reference Standard	Provides benchmark for relative potency calculation	Quality control, assay calibration, comparability studies
Cell-based Assay Systems	Measure functional biological activity	Cytotoxicity, cytokine release, pathway activation
Flow Cytometry	Multiparameter cell surface and intracellular marker analysis	CAR expression, immunophenotyping, viability
PCR/ddPCR	Quantitative measurement of genetic elements	Vector copy number, host cell DNA, viral integration sites
ELISA	Protein quantification and detection	Cytokine secretion, therapeutic protein expression
Multi-omics Platforms	Comprehensive molecular profiling	Genomic, epigenomic, transcriptomic characterization
Colony Forming Unit Assays	Measure proliferative potential and stemness	Hematopoietic stem cell products, progenitor cells

[3] [4] [11]

The experimental design for potency methods must account for inherent bioassay variability through appropriate replication strategies and statistical analysis [3]. A common framework involves Analytical Quality by Design (AQbD) principles, with Design of Experiments (DoE) approaches to identify optimal parameter ranges and test method robustness [3]. Intra-assay variability is often controlled through replication within assay runs, which improves measurement precision and enables statistically driven outlier identification [3].

Challenges and Future Directions

Addressing Variability in Potency Assays

Bioassays used for potency testing typically demonstrate higher variability compared to physicochemical methods due to multiple operational and biological factors [3]. This variability must be carefully characterized and controlled throughout the product lifecycle—during assay development, method validation, and commercial lot testing [3].

Statistical approaches using linear mixed models help estimate different sources of variability and their impact on the accuracy of reportable potency results [3]. The number of assay runs used to derive the reportable potency value represents an important strategy for controlling variability, with averaging across multiple valid runs improving precision and reducing the likelihood of out-of-specification (OOS) results [3].

Emerging Technologies and Approaches

The field of potency testing continues to evolve with advances in analytical technologies and scientific understanding. Emerging approaches include:

• Single-cell multi-omics for correlating cellular characteristics with functional outcomes [4]
• Advanced bioanalytical methods for characterizing complex attributes such as empty versus full capsids in viral vector products [11]
• Novel bioassay platforms that better capture mechanism of action for increasingly complex cell therapy products [4] [12]
• Standardized reference materials and analytical methods to improve inter-laboratory consistency and comparability [12] [11]

The ongoing development of regulatory guidelines reflects the dynamic nature of this field, with recent FDA drafts addressing potency assessment for emerging product categories like genome editing therapies and enhanced CAR T-cell products [1] [4].

Potency testing remains a critical quality attribute and regulatory imperative for cell and gene therapy products, serving as the essential link between product characteristics and clinical performance. The complex nature of these advanced therapies necessitates sophisticated testing strategies that often combine multiple assay formats in an orthogonal approach to fully capture biological activity.

The analysis of FDA-approved products reveals that while viability, count, and expression assays form the foundation of current potency testing practices, functional bioassays and advanced characterization methods are increasingly important for comprehensive product understanding. As the field advances, potency assays continue to evolve from simple correlation-based tests to sophisticated methods grounded in detailed mechanism of action studies employing multi-omics technologies.

Successful potency assay implementation requires early investment in assay development, careful consideration of variability sources, and strategic application of reference standards and orthogonal methods. By addressing these challenges comprehensively, developers can establish robust potency assays that not only meet regulatory requirements but also provide meaningful assurance of product quality, consistency, and clinical performance.

Linking Mechanism of Action (MoA) to Biological Activity

In the field of cell therapy, accurately measuring product potency—the quantitative biological activity linked to the intended therapeutic effect—is a fundamental regulatory and manufacturing requirement [13] [14]. The development of robust potency assays remains intrinsically dependent on a thorough understanding of the therapy's Mechanism of Action (MoA), defined as the specific biochemical interactions through which a product produces its pharmacological effect [15]. For complex living medicines like CAR T-cells, the MoA encompasses a multifaceted sequence of events: specific antigen recognition on target cells, immune activation, proliferation, and execution of cytotoxic functions, followed by persistence and formation of memory populations [4]. As cell therapies evolve, the traditional potency assays, such as IFN-γ release measurements, may no longer capture the full spectrum of biological activity [4] [16]. This guide objectively compares contemporary methodologies for linking MoA to biological activity, providing researchers with experimental data and protocols to advance potency assay development.

Comparative Analysis of MoA-Based Potency Assay Platforms

The following table summarizes key methodologies used for potency assessment based on different aspects of cell therapy MoA, enabling direct comparison of their applications, outputs, and limitations.

Table 1: Comparison of MoA-Based Potency Assay Platforms for Cell Therapies

Assay Platform	Biological Activity Measured	Key Readouts	Therapeutic Context	Advantages	Limitations
Functional Cytotoxicity Assays [17]	Target cell killing capacity, serial killing ability	Luminescence signal from lysed target cells (HiBiT), Loss of viability signal (Bright-Glo)	CAR-T, CAR-NK, TCR-T therapies	Measures direct therapeutic effect; High sensitivity; Real-time kinetics	May not fully predict in vivo persistence
Cytokine Secretion Profiling [4] [17]	T-cell activation, functional polarization	IFN-γ, TNF-α, IL-2 secretion (Lumit Immunoassays, ELISA)	Lot release for FDA-approved CAR-T products; T-cell activation status	Simple, standardized; Correlates with clinical response	Limited scope of MoA capture; Doesn't measure cytotoxicity directly
T-cell Activation Bioassays [17]	CAR/TCR engagement and signaling intensity	NFAT or IL-2 pathway-induced luminescence	CAR/TCR validation; Lentiviral vector potency testing	Specific to receptor signaling; Stability-indicating	Does not incorporate tumor microenvironment factors
Multi-omics Profiling [4]	Genomic, epigenomic, transcriptomic signatures associated with potency	Vector copy number, integration sites, DNA methylation, TCR clonality	CAR-T product profiling; Biomarker discovery for predictive potency	Comprehensive MoA insight; Identifies novel potency markers	Complex data analysis; Not yet standardized for lot release
Phenotypic & Metabolic Assays [16]	Stem-like memory phenotype, metabolic fitness, persistence	T-cell differentiation markers, mitochondrial function, long-term serial killing	Assessing impact of tumor microenvironment on potency	Mimics physiological conditions; Predicts long-term efficacy	Requires specialized culture conditions (e.g., hypoxia)

Experimental Protocols for Key MoA-Linked Potency Assays

Purpose: To quantitatively measure cell therapy-mediated cytotoxicity, a critical MoA for CAR-T and other immune effector cell therapies.

Methodology Details:

• Target Cell Preparation: Engineer tumor cell lines to express a cell membrane-bound HiBiT fusion protein. Seed these cells in culture plates at a density of 10,000-50,000 cells/well.
• Effector Cell Co-culture: Add serially diluted CAR-T cells or other effector cells at various Effector:Target (E:T) ratios. Typical E:T ratios range from 10:1 to 0.1:1.
• Incubation: Incubate co-cultures for 4-72 hours at 37°C, 5% COâ‚‚, depending on the kinetics of killing activity.
• Detection: Add Bio-Glo-NB TCK Reagent containing the LgBiT protein and substrate. Upon target cell lysis (killing), the membrane-bound HiBiT tag is released and binds extracellular LgBiT to form a functional NanoBiT Luciferase enzyme.
• Measurement: Quantify luminescence using a plate luminometer. The signal intensity is directly proportional to the number of lysed target cells.

Data Interpretation: The increase in luminescence signal correlates directly with target cell killing capacity. Data can be expressed as percentage cytotoxicity or plotted as dose-response curves to calculate ECâ‚…â‚€ values for potency comparison.

Purpose: To measure the potency and specificity of CAR or TCR signaling upon antigen engagement.

Methodology Details:

• Effector Cell Engineering: Use engineered T-cell lines (TCR/CD3 Effector Cells) containing an NFAT-responsive luciferase reporter gene. These cells may lack endogenous TCRαβ chains to prevent mispairing (TCRαβ-KO Cells).
• Genetic Modification: Transduce or transfect effector cells with CAR or TCR constructs of interest using lentiviral vectors or other methods.
• Antigen Exposure: Co-culture engineered T-cells with antigen-presenting cells or target cells expressing the cognate antigen. For specificity controls, include antigen-negative or antigen-knockout target cells.
• Activation Period: Incubate for 6-24 hours to allow full activation of signaling pathways and luciferase expression.
• Detection: Add luciferase substrate (e.g., Bio-Glo Luciferase Assay System) and measure luminescence.

Data Interpretation: Luminescence intensity directly correlates with the strength of CAR/TCR signaling. The assay can demonstrate specificity through antigen-knockout controls and can be used for stability testing of viral vectors by monitoring ECâ‚…â‚€ shifts in forced degradation studies.

Purpose: To comprehensively characterize CAR-T products at genomic, epigenomic, and transcriptomic levels for identification of novel potency markers.

Methodology Details:

• Sample Collection: Collect CAR-T cells at multiple manufacturing timepoints: starting material, during expansion, and in final product.
• Genomic Analysis:
  • Vector Copy Number (VCN): Quantify using droplet digital PCR (ddPCR) [4] [18].
  • Integration Sites: Map using next-generation sequencing pipelines (INSPIIRED or EpiVIA) [4].
  • TCR Repertoire: Profile using bulk or single-cell TCR sequencing (TCR-seq) [4].
• Epigenomic Analysis:
  • Analyze DNA methylation profiles using bisulfite sequencing.
  • Assess chromatin accessibility via ATAC-seq.
• Transcriptomic Analysis:
  • Perform bulk or single-cell RNA sequencing to identify gene expression signatures associated with positive clinical outcomes.
• Data Integration: Correlate multi-omics profiles with functional potency measures and clinical outcomes to identify predictive potency biomarkers.

Data Interpretation: Specific epigenetic signatures (e.g., 18 distinct epigenetic loci identified in CD19 CAR-T products) and TCR clonal diversity metrics can be correlated with complete response rates and survival outcomes [4].

Visualizing the Relationship Between MoA and Potency Assays

Diagram 1: MoA to Potency Assay Relationship. This diagram illustrates how different aspects of a therapy's Mechanism of Action inform the development of specific potency assay platforms, which collectively predict clinical response.

Essential Research Reagents for MoA-Focused Potency Testing

Table 2: Key Research Reagent Solutions for Potency Assay Development

Reagent / Assay System	Primary Function	Application in MoA-Linked Potency
Lumit Cytokine Immunoassays [17]	Detection of cytokine secretion (IFN-γ, TNF-α, IL-2)	Measures T-cell activation; Homogeneous, no-wash alternative to ELISA
HiBiT Target Cell Killing Bioassay [17]	Quantification of target cell lysis by effector cells	Direct measurement of cytotoxic MoA; Gain-of-signal approach
Bright-Glo / One-Glo Luciferase Assay Systems [17]	Measurement of viability in engineered tumor cells	Loss-of-signal cytotoxicity measurement
T-cell Activation Bioassay (NFAT/IL-2) [17]	Reporter-based detection of TCR/CAR signaling	Validates receptor function and specificity
T-cell Activation Bioassay (TCRαβ-KO) [17]	Prevents endogenous TCR mispairing	Critical for accurate TCR-T therapy potency assessment
ddPCR Technology [4] [18]	Absolute quantification of vector copy number	Genomic component of multi-omics potency assessment
Single-cell RNA-seq Platforms [4]	Transcriptomic profiling at single-cell resolution	Identifies subpopulations correlated with clinical potency
AVATAR Cell Culture Platform [16]	Mimics tumor microenvironment (hypoxic conditions)	Assesses potency under physiologically relevant conditions

The evolving landscape of cell therapy demands increasingly sophisticated approaches to potency assessment that fully capture the complexity of therapeutic MoA. While traditional cytokine release assays remain valuable for lot release of approved products [4], emerging methodologies—particularly functional killing assays and multi-omics profiling—provide deeper insights into the critical biological activities that drive clinical efficacy [4] [16] [17]. The most advanced approaches integrate multiple assay platforms to create a comprehensive potency matrix that reflects different aspects of the MoA, from immediate cytotoxic function to long-term persistence potential. As research continues to identify novel biomarkers and functional correlates of clinical response, potency assays will increasingly evolve from simple quality control metrics to predictive tools that inform both manufacturing and clinical application, ultimately strengthening the bridge between mechanistic understanding and therapeutic performance.

Common Challenges and Development Delays in Potency Testing

Potency testing, defined as the quantitative measure of a biological product's specific ability or capacity to effect a given result, represents one of the most significant challenges in the development of cell and gene therapies [19]. For Advanced Therapy Medicinal Products (ATMPs), potency assays are not merely regulatory checkboxes but fundamental tools that ensure manufacturing consistency, product quality, and ultimately, patient safety and efficacy [5]. The complexity of these living medicines introduces unique challenges that routinely delay development timelines and market entry, creating critical bottlenecks in delivering transformative treatments to patients.

The stakes are exceptionally high. According to regulatory analyses, nearly 50% of ATMP applications encounter potency-related issues during assessment, often resulting in significant delays [20]. Former FDA CBER Director Peter Marks has noted that "potency-related problems have been among the most common reasons for delayed approvals in the field" [20]. These delays carry profound consequences, with each month of postponement potentially costing $5–15 million in lost revenue and, more importantly, accumulating thousands of patient-days without access to potentially life-saving therapies [20].

Common Potency Testing Challenges

Biological Complexity and Variable Nature of Living Products

Unlike conventional pharmaceuticals, cell therapies are characterized by exceptional complexity and heterogeneity. These living products exhibit higher intrinsic variability due to donor-to-donor differences, complex mechanisms of action (MoA), and limited stability profiles [21] [19]. This biological variability directly translates into challenges in developing robust potency assays that can consistently measure product quality across multiple batches.

For cellular products, basic attributes like viability and cell phenotype, while important, are insufficient to fully address biological activity [19]. A comprehensive understanding of the product's functional characteristics is required, which often demands a matrix of tests rather than a single assay [8]. The living nature of these products means they continue to evolve and differentiate, making it difficult to establish static quality standards that remain relevant throughout the product's shelf life and after administration.

Technical and Methodological Hurdles

Potency assays for ATMPs face substantial technical challenges that contribute to development delays:

• High assay variability: Bioassays typically demonstrate higher variability compared to physicochemical methods due to multiple operational and biological factors [3]. This variability complicates validation and necessitates complex statistical approaches to ensure reliability.

• Lack of standardized methods: Unlike compendial methods used for traditional drugs, potency assays for ATMPs are typically developed from "scratch" and cannot benefit from multi-company improvement across years of international standardization [3].

• Complexity of relative potency measurements: Most potency assays report results as % Relative Potency (%RP) derived from modeled fits of dose-response data, requiring rigorous parallelism testing between reference standards and test samples [3]. The fundamental assumption of parallelism must be met for meaningful derivation of %RP, adding another layer of methodological complexity.

Regulatory and Strategic Challenges

Navigating the regulatory landscape for potency testing presents additional obstacles:

• Differing regulatory expectations: While the US FDA generally expects a quantitative functional potency assay for product release, EU regulations may allow validated surrogate assays for release testing when a functional assay is available for characterization and correlation can be demonstrated [19]. This discrepancy can complicate global development strategies.

• Late consideration in development pipelines: Potency testing is often treated as an afterthought rather than being integrated into development strategies from day one [20]. This delayed focus creates downstream bottlenecks when sponsors must rapidly develop and validate assays for late-stage clinical trials and marketing applications.

• Matrix approach requirements: For products with multiple mechanisms of action, regulators may expect a potency assay matrix rather than a single test [8]. Developing, validating, and maintaining multiple complex assays significantly extends development timelines.

Quantitative Analysis of Current Practices

Potency Test Utilization in Approved Products

Analysis of the 31 US FDA-approved cell therapy products reveals critical patterns in potency testing implementation. A total of 104 potency tests have been used for these products, with an average of 3.4 tests per product (standard deviation 2.0) [8]. Notably, 32% of these tests are redacted in public documentation, indicating their proprietary nature and potential competitive advantage [8].

Table 1: Distribution of Potency Test Types Across FDA-Approved Cell Therapy Products

Test Category	Number of Tests	Percentage of Non-Redacted Tests	Example Applications
Viability and Count	37	52%	Cell viability, viable cell count
Expression	19	27%	CAR expression, surface markers
Bioassays	7	7%	Cytotoxicity, cytokine release
Genetic Modification	6	9%	Vector copy number, transgene expression
Histology	2	3%	Tissue morphology, structural integrity

Source: Adapted from analysis of 31 US FDA-approved cell therapy products [8]

The data reveals that "Viability and count" and "Expression" assays are most frequently employed, often together in the same product (16 of 31 CTPs) [8]. This combination suggests that while basic quality attributes are necessary, they are supplemented with product-specific characterisation to address unique mechanisms of action.

Impact of Product Class on Testing Complexity

The complexity of potency testing varies significantly by product class, with more technologically advanced therapies generally requiring more sophisticated testing approaches:

Table 2: Potency Test Frequency by Cell Therapy Product Class

Product Class	Number of Products	Average Number of Potency Tests	Standard Deviation
Hematopoietic Stem Cell-Cord Blood	5	4.4	0.7
CAR T-cell Products	7	1.9	0.9
Tissue Engineered Products	5	1.8	1.1
All Approved CTPs	31	3.4	2.0

Source: Adapted from analysis of FDA-approved cellular therapies [8]

Interestingly, the data does not show a significant increase in the number of potency tests per product over time, suggesting that scientific advances have not necessarily translated into more complex testing requirements but rather more sophisticated individual assays [8].

Detailed Experimental Approaches

CAR T-cell Potency Assessment

CAR T-cell products present unique potency testing challenges due to their complex mechanism of action involving target recognition, T-cell activation, proliferation, and ultimately, cytotoxic activity. Conventional potency assessments for FDA-approved CAR T-cell products typically measure IFN-γ release in response to target cells, alongside cell viability and product-specific attributes like CAR expression [4].

An innovative flow cytometry-based approach has been developed that measures CD69 expression as a specific indicator of CAR functionality [22]. This quantitative method correlates with traditional cytotoxicity measurements while offering improved precision and reduced variability:

CAR T-cell Potency Assay Workflow

The CD69-based assay demonstrates how surrogate markers of T-cell activation can provide robust, quantitative potency measurements that are directly linked to the product's biological activity while being more amenable to quality control environments than complex functional assays [22].

Mesenchymal Stromal Cell (MSC) Potency Assessment

MSCs present distinct challenges for potency testing due to their multiple immunomodulatory mechanisms and donor-to-donor variability. A therapeutically relevant potency assay for ABCB5+ MSCs was developed to quantify anti-inflammatory capacity through IL-1RA secretion in an M1 macrophage-dominated inflammatory environment [21].

Table 3: Research Reagent Solutions for MSC Potency Testing

Reagent/Cell Line	Function in Assay	Application Details
THP-1 Monocyte Cell Line	Differentiation into M1 macrophages	Creates inflammatory environment mimicking in vivo conditions
Phorbol 12-Myristate 13-Acetate (PMA)	THP-1 differentiation inducer	150 nmol/ml for 48 hours
Recombinant Human IFN-γ	M1 polarization stimulus	50 IU/ml at co-culture start and 24 hours
LPS from E. coli O111:B4	M1 polarization enhancement	20 ng/ml at 24 hours
Anti-CD36 Antibody	Macrophage differentiation marker	Flow cytometry confirmation (>50% expression)
Anti-CD80 Antibody	M1 macrophage marker	Flow cytometry confirmation (>50% expression)
Human IL-1RA/IL-1F3 Quantikine ELISA Kit	IL-1RA quantification	Validated per EMA/FDA guidelines, LLOQ: 125 pg/ml

Source: Adapted from PMC11065629 [21]

The experimental protocol involves:

• Macrophage Differentiation: THP-1 cells are differentiated using PMA-containing medium for 48 hours
• Co-culture Establishment: Differentiated macrophages are co-cultured with ABCB5+ MSCs at optimized ratios
• M1 Polarization: IFN-γ and LPS are added to drive and maintain M1 polarization
• IL-1RA Quantification: Supernatants are collected after 48 hours for IL-1RA measurement by validated ELISA

This assay successfully addresses the challenge of modeling complex in vivo interactions in a controlled in vitro system, demonstrating therapeutic relevance for conditions dominated by innate immune activation [21].

Consequences of Testing Deficiencies

Impact on Patient Access and Therapeutic Development

Delays in potency assay development directly impact patient access to advanced therapies. In conditions like relapsed large B-cell lymphoma, clinical data suggest that 20-25% of patients may lose performance status each month without effective therapy, potentially disqualifying them from receiving CAR-T or other advanced treatments [20]. The cumulative impact of delays can be quantified through patient-day loss metrics, where a one-month delay for a batch treating 100 patients equals 3,000 patient-days without therapy—more than eight years of cumulative lost treatment time across the waiting population [20].

Economic Implications

The financial consequences of potency testing delays are substantial, with each month of delay for a high-value therapy estimated to cost $5-15 million in lost revenue [20]. These figures do not include additional costs from manufacturing holds, supply chain disruptions, or opportunity costs from missed patient enrollment in clinical trials. For small biotech companies and academic developers, these economic pressures can threaten the viability of promising therapies long before they reach patients.

Emerging Solutions and Future Directions

Advanced Analytical Technologies

The field is rapidly evolving with new technologies offering potential solutions to longstanding potency testing challenges:

• Multi-omics approaches: Genomics, epigenomics, transcriptomics, proteomics, and metabolomics at both bulk and single-cell resolution provide comprehensive molecular profiling that enhances understanding of CAR T-cell function [4]. These technologies enable identification of novel potency markers beyond traditional functional assays.

• Advanced genomic profiling: Vector copy number (VCN) quantification, vector integration site analysis, and T-cell receptor (TCR) repertoire sequencing provide critical safety and efficacy insights [4]. Tools like the INSPIIRED and EpiVIA pipelines enable detection of integration events at bulk and single-cell resolution.

• Epigenetic profiling: DNA methylation analysis has identified distinct epigenetic loci associated with complete response, event-free survival, and overall survival post-infusion [4]. These epigenetic signatures may serve as predictive potency markers.

Strategic and Regulatory Advancements

Beyond technological innovations, strategic approaches are emerging to address potency testing challenges:

• Early integration: Incorporating potency assay strategy from day one of product development, with assays designed around the therapy's mechanism of action [20]

• Proactive regulatory engagement: Early alignment with regulatory agencies on potency testing strategies to reduce the risk of rework or rejection [20]

• Platform approaches: Leveraging experience across similar manufacturing processes and potency method types to establish platform acceptance criteria, particularly for early-stage development [3]

The ongoing development of standardized reference materials and assay platforms by organizations like the National Institute of Standards and Technology (NIST) promises to further reduce variability and improve reproducibility across the industry [23].

Potency testing remains a critical challenge in the development of cell and gene therapies, with significant implications for both patient access and economic viability. The biological complexity of these living medicines, combined with technical methodological hurdles and evolving regulatory expectations, creates a landscape where delays are common and consequences are substantial.

However, emerging technologies and strategic approaches offer promising paths forward. From innovative assay designs that better model disease-specific microenvironments to advanced analytical methods that provide deeper product characterization, the field is developing increasingly sophisticated tools to address these challenges. The key to success lies in early and strategic focus on potency testing, integration of novel technologies, and collaborative engagement with regulatory agencies throughout the development process.

As the field continues to mature, the evolution of potency testing strategies will play a pivotal role in realizing the full potential of advanced therapies, ensuring that these transformative treatments can reach patients in a timely, safe, and effective manner.

Implementing Potency Assays: From Design Principles to Real-World Applications

The development of robust potency assays is a critical regulatory requirement that ensures the quality, safety, and efficacy of cell therapy products throughout their lifecycle from research to clinical application [18]. These assays are designed to measure the biological activity of a product based on its specific mechanism of action (MoA) and are essential for lot-release testing, stability studies, and demonstrating product consistency [4] [18]. For cell therapies, particularly those involving complex living cells, potency represents a composite of multiple functional activities rather than a single attribute. The evolving landscape of FDA-approved cell therapies—which includes CAR-T cells, hematopoietic progenitor cells (HPCs), and other cellular products—demands equally advanced analytical approaches to fully characterize their therapeutic potential [4] [24].

This analysis examines the methodological frameworks employed for 31 FDA-approved cell therapies, with a focus on the experimental workflows and analytical techniques that define modern potency testing. As the field advances with new technologies and manufacturing strategies, conventional potency assays may not fully capture product complexity, driving the development of tailored potency assays that incorporate multi-omics profiling and functional assessments [4]. This review provides researchers and drug development professionals with a comprehensive comparison of analytical methods, detailed experimental protocols, and essential research tools that form the foundation of cell therapy characterization.

The U.S. Food and Drug Administration has approved numerous cell-based therapies for a range of conditions, primarily in oncology, hematology, and regenerative medicine. These products can be broadly categorized into CAR-T cell therapies, umbilical cord blood-derived HPCs, and other cell therapies with diverse mechanisms of action [25] [26]. The following table summarizes 31 approved cell therapies, their indications, and key characteristics.

Table 1: FDA-Approved Cell Therapies and Their Key Characteristics

Product Name	Manufacturer	Year Approved	Therapeutic Category	Indication
ABECMA (idecabtagene vicleucel)	Celgene Corporation	2021	CAR-T Cell Therapy	Refractory Multiple Myeloma
AMTAGVI (lifileucel)	Iovance Biotherapeutics	2024	Tumor-Infiltrating Lymphocytes (TIL)	Unresectable or Metastatic Melanoma
AUCATZYL (obecabtagene autoleucel)	Autolus Limited	2024	CAR-T Cell Therapy	B-cell Acute Lymphoblastic Leukemia (B-ALL)
BREYANZI (lisocabtagene maraleucel)	Juno Therapeutics	2021	CAR-T Cell Therapy	Relapsed/Refractory Large B-cell Lymphoma
CARVYKTI (ciltacabtagene autoleucel)	Janssen Biotech	2022	CAR-T Cell Therapy	Relapsed/Refractory Multiple Myeloma
CASGEVY (exagamglogene autotemcel)	Vertex Pharmaceuticals	2023	Gene-Edited HSC	Sickle Cell Disease
CLEVECORD (HPC Cord Blood)	Cleveland Cord Blood Center	2016	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
Ducord (HPC Cord Blood)	Duke University School of Medicine	2012	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
GINTUIT (Allogeneic Cultured Keratinocytes and Fibroblasts)	Organogenesis Incorporated	2012	Cellularized Scaffold	Mucogingival Conditions
HEMACORD (HPC, Cord Blood)	New York Blood Center	2011	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
HPC, Cord Blood - Bloodworks	Bloodworks	2016	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
HPC, Cord Blood - Clinimmune Labs	Clinimmune Labs, University of Colorado	2012	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
HPC, Cord Blood - LifeSouth	LifeSouth Community Blood Centers	2016	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
HPC, Cord Blood - MD Anderson	MD Anderson Cord Blood Bank	2018	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
KYMRIAH (tisagenlecleucel)	Novartis Pharmaceuticals	2017	CAR-T Cell Therapy	B-cell Acute Lymphoblastic Leukemia (B-ALL)
LANTIDRA (donislecel)	CellTrans Inc.	2023	Allogeneic Pancreatic Islet Cells	Type 1 Diabetes
LAVIV (Azficel-T)	Fibrocell Technologies	2011	Autologous Fibroblasts	Nasolabial Fold Wrinkles
LYFGENIA (lovotibeglogene autotemcel)	bluebird bio, Inc.	2023	Gene-Modified HSC	Sickle Cell Disease
MACI (Autologous Cultured Chondrocytes)	Vericel Corp.	2016	Autologous Chondrocytes	Cartilage Defects
OMISIRGE (omidubicel-onlv)	Gamida Cell Ltd.	2023	Nicotinamide-Modified HPC	Hematopoietic Reconstitution
PROVENGE (sipuleucel-T)	Dendreon Corp.	2010	Autologous Cellular Immunotherapy	Prostate Cancer
REGENECYTE (HPC, Cord Blood)	StemCyte, Inc.	2008	Umbilical Cord Blood HPC	Hematopoietic Reconstitution
RETHYMIC (Allogeneic Processed Thymus Tissue)	Enzyvant Therapeutics GmbH	2021	Thymus Tissue	Congenital Athymia
RYONCIL (remestemcel-L)	Mesoblast, Inc.	2024	Allogeneic Mesenchymal Stem Cells	Pediatric Graft vs. Host Disease
STRATAGRAFT (Allogeneic Cultured Keratinocytes and Dermal Fibroblasts)	Stratatech Corporation	2021	Cellularized Scaffold	Thermal Burns
SYMVESS (Acellular Tissue Engineered Vessel)	Humacyte Global, Inc.	2024	Tissue-Engineered Vessel	Vascular Repair
TECARTUS (brexucabtagene autoleucel)	Kite Pharma, Inc.	2020	CAR-T Cell Therapy	Relapsed/Refractory Mantle Cell Lymphoma
TECELRA (afamitresgene autoleucel)	Adaptimmune LLC	2024	Engine T-cell Therapy	Synovial Sarcoma, Myxoid Round Cell Liposarcoma
YESCARTA (axicabtagene ciloleucel)	Kite Pharma, Inc.	2017	CAR-T Cell Therapy	Relapsed/Refractory Large B-cell Lymphoma
ZYNTEGLO (betibeglogene autotemcel)	bluebird bio, Inc.	2022	Gene-Modified HSC	β-thalassemia

The diversity of these approved products highlights the expanding scope of cell therapy and underscores the need for equally diverse analytical methods to ensure their safety and efficacy. CAR-T cell therapies dominate the engineered cell therapy landscape, with seven approved products targeting hematological malignancies [26]. Umbilical cord blood-derived HPCs represent the largest category of approved cell therapies, with nine products approved for hematopoietic reconstitution [26]. More recent approvals include advanced engineered T-cell therapies like TECELRA and non-traditional cellular products like SYMVESS, reflecting the innovative approaches entering the clinical landscape.

Analytical Methodologies for Cell Therapy Characterization

Established Potency Assay Platforms

The potency assessment of cell therapies relies on a matrix of complementary assays that evaluate different aspects of biological activity. For FDA-approved CAR-T cell products, potency is primarily assessed by measuring IFN-γ release in response to target cells, along with other factors such as cell viability, and product-specific attributes including CAR expression levels [4]. These established methods form the foundation of lot-release testing for commercial products.

Table 2: Core Potency Assay Methods for Cell Therapies

Assay Category	Measured Parameters	Common Techniques	Primary Applications
Functional Potency	Cytokine secretion (IFN-γ, TNF-α, IL-2), Cytotoxic activity	ELISA, ELISpot, Flow cytometry, Real-time cell analysis	CAR-T cells, TIL therapies, Activated cell products
Phenotypic Characterization	Surface marker expression, Cell population distribution	Flow cytometry, Immunofluorescence	All cell therapies, especially CAR-T and HPC products
Genomic Analysis	Vector copy number (VCN), Transgene integration, TCR repertoire	ddPCR, qPCR, Next-generation sequencing	Genetically modified cells (CAR-T, gene-edited therapies)
Viability and Expansion Capacity	Cell count, Viability, Proliferation rate	Trypan blue exclusion, Flow cytometry, Metabolic assays	All living cell therapy products

The functional potency assays are particularly crucial as they measure the direct biological activity of the therapeutic product. For CAR-T cells, this includes evaluating their ability to recognize target antigens, activate appropriate signaling pathways, and execute effector functions including cytokine release and direct cytotoxicity [4]. These functional assessments often employ co-culture systems with antigen-positive target cells, with measurements of activation markers, cytokine production, and target cell killing serving as key potency indicators.

Advanced Multi-Omics Profiling Approaches

Recent advances in analytical technologies have enabled more comprehensive characterization of cell therapy products through multi-omics approaches. These methods provide deeper insights into product characteristics that correlate with clinical efficacy but may not be fully captured by conventional potency assays [4].

Genomic profiling of cell therapy products has expanded beyond mandatory vector copy number (VCN) quantification to include vector integration site analysis and T cell receptor (TCR) repertoire sequencing [4]. The development of robust pipelines for detecting viral integration events, such as the INSPIIRED and EpiVIA pipelines, has improved our ability to monitor insertional mutagenesis risks and clonal dynamics [4]. TCR repertoire assessment through immunogenomic analyses provides valuable information about the diversity and clonality of CAR-T cell products, which may influence treatment outcomes [4].

Epigenomic profiling through DNA methylation analysis and chromatin accessibility mapping has emerged as a powerful approach for understanding CAR T-cell differentiation states, which are critically important for therapeutic efficacy [4]. Carlos et al. analyzed DNA methylation profiles in 114 CD19 CAR T-cell products and identified 18 distinct epigenetic loci associated with complete response, event-free survival, and overall survival post-infusion [4]. These epigenetic markers provide insights beyond transcriptomic profiling alone and may guide the development of next-generation potency assays.

Transcriptomic, proteomic, and metabolomic profiling at both bulk and single-cell resolution further enhance our understanding of CAR T-cell function at the molecular level [4]. Single-cell RNA sequencing enables the identification of distinct transcriptional phenotypes within infusion products, while proteomic analyses reveal protein expression patterns associated with T-cell differentiation states. Metabolomic assessments provide insights into the energy metabolism and metabolic fitness of cell therapy products, which can influence their in vivo persistence and functionality.

Experimental Protocols for Key Potency Assays

CAR T-Cell Functional Potency Assessment

The functional potency of CAR T-cell products is typically evaluated through a comprehensive protocol that assesses multiple aspects of their biological activity. The following protocol outlines the key steps for evaluating CAR T-cell potency:

• CAR T-Cell and Target Cell Preparation

  • Thaw CAR T-cell products and maintain in appropriate culture medium supplemented with IL-2 (50-100 IU/mL)
  • Culture target cells (antigen-positive and antigen-negative control lines) to 70-80% confluence
  • Determine cell counts and viability using trypan blue exclusion or automated cell counters

• Co-Culture Setup for Functional Assessment

  • Seed target cells in 96-well plates at a density of 1×10^4 cells per well
  • Add CAR T-cells at effector-to-target (E:T) ratios ranging from 1:1 to 10:1
  • Include appropriate controls (CAR T-cells alone, target cells alone, antigen-negative target cells)
  • Incubate at 37°C, 5% CO2 for 18-24 hours

• Cytokine Secretion Analysis

  • Collect supernatant from co-culture wells
  • Measure IFN-γ, TNF-α, and IL-2 concentrations using ELISA or multiplex immunoassays
  • Compare cytokine levels across different E:T ratios and against control conditions

• Cytotoxic Activity Assessment

  • Measure target cell killing using real-time cell analysis or flow cytometry-based cytotoxicity assays
  • For flow cytometry-based approaches, stain target cells with CFSE prior to co-culture
  • Add viability dye (e.g., propidium iodide) before acquisition
  • Calculate specific lysis using the formula: ( experimental mortality - spontaneous mortality ) / (100 - spontaneous mortality ) × 100

• Activation Marker Expression

  • Harvest cells from co-culture wells after 18-24 hours
  • Stain with antibodies against CD69, CD25, and activation-specific isoforms of CD45
  • Analyze by flow cytometry to determine activation status

This protocol provides a comprehensive assessment of CAR T-cell functional potency, capturing multiple mechanisms of action that contribute to their therapeutic efficacy. The inclusion of antigen-negative controls ensures the specificity of the observed responses, while the use of multiple E:T ratios provides information about dose-response relationships.

Advanced Functional Assay Mimicking Tumor Microenvironment

To address limitations of conventional cytokine secretion assays, which lack direct functional information on target cell status and cannot predict long-term persistence, researchers have developed more sophisticated assay systems. The following protocol describes a functional assay using the AVATAR platform to mimic the tumor microenvironment:

• CAR T-Cell Manufacturing under Physiologic Conditions

  • Culture CAR T-cells under hypoxic (2-5% O2) and hyperbaric conditions mimicking the tumor microenvironment
  • Maintain cells in specialized bioreactors that allow control of oxygen tension and pressure
  • Monitor cell growth and viability throughout the manufacturing process

• Stemness Phenotype Assessment

  • Analyze CAR T-cells for stem cell memory markers (CD62L, CCR7, CD95)
  • Perform transcriptional profiling for genes associated with stem-like phenotype (TCF7, LEF1)
  • Evaluate mitochondrial fitness through seahorse analysis or flow cytometry with mitochondrial dyes

• Serial Killing Capacity Evaluation

  • Set up repeated challenge assays with fresh target cells added at regular intervals (every 48-72 hours)
  • Monitor CAR T-cell expansion and persistence through flow cytometry counting
  • Assess accumulation of exhaustion markers (PD-1, TIM-3, LAG-3) over multiple cycles

• Long-term Functional Persistence Assessment

  • Culture CAR T-cells with target cells for extended periods (14-21 days)
  • Periodically sample to evaluate maintenance of cytotoxic function
  • Analyze T-cell differentiation status through CD45RA, CD62L, and CD127 expression

This advanced assay system has demonstrated the ability to identify CAR T-cell products with enhanced stem-like phenotypes, improved tumor killing capacity, and better long-term serial killing activity compared to standard manufacturing approaches [16]. These functional assessments provide more accurate predictions of in vivo potency and persistence, addressing a critical gap in conventional potency testing.

Visualization of Key Analytical Workflows

CAR T-Cell Potency Assessment Workflow

CAR T-Cell Potency Assessment Workflow: This diagram illustrates the comprehensive matrix of assays required to fully characterize CAR T-cell potency, including functional, genomic, phenotypic, and multi-omics approaches.

Multi-Omics Profiling in Cell Therapy Characterization

Multi-Omics Profiling in Cell Therapy: This diagram outlines the comprehensive multi-omics approaches used to characterize cell therapy products at genomic, epigenomic, transcriptomic, proteomic, and metabolomic levels.

The Scientist's Toolkit: Essential Research Reagents and Platforms

The characterization of cell therapies requires specialized reagents, instruments, and platforms that enable comprehensive assessment of product quality, potency, and functionality. The following table details key research solutions used in the development and analysis of cell therapy products.

Table 3: Essential Research Reagents and Platforms for Cell Therapy Analysis

Category	Product/Platform	Primary Function	Application in Cell Therapy
Vector Quantification	Droplet Digital PCR (ddPCR) Systems	Absolute quantification of vector copy number (VCN)	Quality control for genetically modified cell therapies [4]
Cell Analysis	Flow Cytometers with 10+ colors	Multiparameter phenotypic and functional analysis	CAR expression, immunophenotyping, activation status [4]
Functional Assay	AVATAR Platform (Xcellbio)	Mimics tumor microenvironment conditions	Assessment of potency and persistence under physiologic stress [16]
Genomic Analysis	Next-generation Sequencers	TCR repertoire, integration site analysis	Product characterization and safety assessment [4]
Cytokine Detection	Multiplex Immunoassay Systems	Simultaneous measurement of multiple cytokines	Potency assessment through cytokine release profiling [4]
Cell Culture	GMP-grade Cytokines (IL-2, IL-7, IL-15)	Maintenance and expansion of T-cell products	Manufacturing and potency maintenance [4]
Metabolic Analysis	Seahorse Analyzers	Real-time assessment of metabolic function	Evaluation of metabolic fitness and persistence potential [4]
Single-cell Analysis	Single-cell RNA Sequencing Platforms	Transcriptomic profiling at single-cell resolution	Identification of cell subpopulations and differentiation states [4]
Moclobemide-d8	Moclobemide-d8 Stable Isotope	Moclobemide-d8 is a high-purity internal standard for quantitative LC-MS/MS analysis in ADME/DMPK studies. For Research Use Only. Not for human or veterinary use.	Bench Chemicals
Butoconazole-d5 (nitrate)	Butoconazole-d5 (nitrate), MF:C19H18Cl3N3O3S, MW:479.8 g/mol	Chemical Reagent	Bench Chemicals

These tools enable the comprehensive characterization required for regulatory compliance and product understanding. As the field advances, integration of these platforms into standardized workflows is essential for ensuring consistent product quality and facilitating comparability across development stages.

The landscape of analytical methods for FDA-approved cell therapies continues to evolve in parallel with product innovation. The transition from conventional potency assays toward multi-parametric assessment strategies reflects the growing recognition that therapeutic efficacy emerges from multiple biological attributes rather than a single functional readout. The integration of advanced omics technologies with functional assays provides unprecedented insights into the characteristics that correlate with clinical success, enabling more predictive potency assessment.

Future developments in cell therapy analytics will likely focus on real-time potency monitoring, advanced biosensors for continuous functional assessment, and artificial intelligence-driven predictive modeling that integrates multi-omics data with clinical outcomes. Additionally, as allogeneic ("off-the-shelf") cell therapies progress through clinical development, analytical methods must adapt to address new quality attributes specific to these products, including alloreactivity potential and persistence dynamics. The continued refinement of potency assays remains essential for realizing the full therapeutic potential of cell-based medicines and ensuring their consistent manufacturing, safety, and efficacy for patients.

In the development and quality control of Cell Therapy Products (CTPs), potency assays are critical release tests required by regulatory bodies like the US FDA. Their primary function is to ensure that a product can achieve its intended biological effect, assess manufacturing consistency, and evaluate product stability [8]. A review of the 31 US FDA-approved CTPs reveals that developers employ a multi-faceted testing strategy, utilizing an average of 3.4 potency tests per product to comprehensively assure product quality and function [8]. These tests are broadly categorized into five main types: "Viability and count," "Expression," "Bioassays," "Genetic modification," and "Histology" [8]. This guide provides a comparative analysis of the four most prominent assay categories—Viability, Expression, Bioassays, and Genetic Modification—offering researchers a framework for selecting and implementing appropriate assays for their CTPs.

Comparative Analysis of Key Assay Categories

An analysis of regulatory submissions for approved CTPs provides critical insights into the real-world application and importance of different assay types. The table below summarizes the adoption frequency and core purpose of each key assay category.

Table 1: Adoption and Purpose of Key Assay Categories in FDA-Approved Cell Therapies

Assay Category	Adoption in FDA CTPs	Primary Measurement Purpose
Viability and Count	19 of 31 CTPs (61%) [8]	Assesses cellular health, survival, and quantity [27] [8].
Expression	20 of 31 CTPs (65%) [8]	Measures presence and levels of specific genes or proteins (e.g., CAR expression) [8].
Bioassay	7 of 31 CTPs (23%)* [8]	Quantifies a drug's ability to modify a biological process, confirming mechanism of action [8] [28].
Genetic Modification	6 of 31 CTPs (19%) [8]	Confirms specific genetic alterations, such as gene insertion or editing [8].

• Note: Due to data redactions in regulatory documents, as many as 24 (77%) CTPs could potentially use a bioassay, making its true adoption rate unclear [8].

The data shows that Expression and Viability assays are the most commonly cited non-redacted tests, used by over 60% of approved therapies. Furthermore, these two assays are frequently used in combination, occurring together for 16 of the 31 CTPs (52%), highlighting the value of a multi-attribute approach to potency testing [8].

Detailed Category Breakdown & Experimental Protocols

Viability and Count Assays

Cell viability assays are fundamental tools used to determine the percentage of live cells in a population and the total cell count. They are crucial for confirming that a sufficient number of viable, functional cells are present in the final product to elicit a therapeutic effect [27] [8].

• Typical Experimental Protocol: A common method is the use of metabolic assays like MTT or resazurin. These assays rely on the reduction of a reagent by metabolically active cells, producing a measurable signal change (color, fluorescence) that correlates with the number of viable cells [27].
  • Sample Preparation: A single-cell suspension of the CTP is prepared.
  • Reagent Addition: A reagent like resazurin is added to the cell culture.
  • Incubation: The cells are incubated for a defined period (e.g., 1-4 hours) to allow viable cells to convert resazurin into fluorescent resorufin.
  • Signal Measurement: The fluorescence is measured using a plate reader.
  • Data Analysis: The signal intensity is compared to a standard curve of known cell concentrations to calculate the viable cell count and percentage.

Expression Assays

Expression assays measure the presence and quantity of specific gene or protein markers that are critical to the CTP's identity and function. For example, in CAR T-cell therapies, measuring the percentage of cells expressing the chimeric antigen receptor is a direct measure of product identity and potency [8].

• Experimental Protocol (Flow Cytometry for Protein Expression): Flow cytometry is a widely used technique for quantifying protein expression in individual cells.
  • Cell Staining: Cells are stained with fluorescently labeled antibodies specific to the target protein (e.g., an anti-CAR antibody).
  • Incubation & Washing: Cells are incubated to allow antibody binding, then washed to remove unbound antibody.
  • Data Acquisition: Cells are passed single-file through a flow cytometer, which uses lasers to detect the fluorescent signal on each cell.
  • Gating and Analysis: Data analysis software is used to gate on the cell population of interest and determine the percentage of cells that are positive for the marker and the mean fluorescence intensity, which can correlate with expression level.

Bioassays

Bioassays are considered the gold standard for potency testing as they quantitatively measure a drug's ability to modify a biological process, providing direct insight into its mechanism of action [28]. Unlike other assays, they report on the collective functional output of the living cells or biological product.

• Experimental Protocol (Cytotoxicity Bioassay for CAR T Cells): This assay measures the ability of CAR T cells to kill target cells expressing the relevant antigen.
  • Target Cell Preparation: Target cells (e.g., a tumor cell line) are labeled with a fluorescent dye.
  • Co-Culture: The labeled target cells are co-cultured with the CAR T-cell product at various effector-to-target (E:T) ratios.
  • Incubation: The cells are incubated for a defined period (e.g., 4-24 hours) to allow for killing.
  • Signal Measurement: The supernatant is measured for fluorescence, which is released upon target cell death. Alternatively, luciferase-based assays can be used where target cell viability is measured via luminescence.
  • Data Analysis: Percentage cytotoxicity is calculated based on the signal compared to controls (target cells alone for maximum lysis, and with medium only for spontaneous lysis). The results demonstrate the lot-to-lot functional potency of the CTP.

Genetic Modification Assays

These assays confirm the successful introduction, deletion, or editing of specific genetic sequences in a CTP. This is essential for genetically modified therapies to ensure the correct genetic construct is present and functional [8].

• Experimental Protocol (Digital PCR for Vector Copy Number): Digital PCR (dPCR) provides an absolute count of target DNA molecules without the need for a standard curve, making it highly precise for quantifying genetic modifications.
  • Sample Digestion: Genomic DNA is extracted from the CTP and digested.
  • Partitioning: The DNA sample is partitioned into thousands of individual reactions, so that each contains zero, one, or a few DNA molecules.
  • Endpoint PCR: Each partition undergoes a PCR reaction with fluorescent probes specific to the transgene (e.g., the CAR) and a reference gene.
  • Data Analysis: The dPCR instrument counts the number of positive (fluorescent) partitions for each target. Using Poisson statistics, it calculates the absolute number of transgene copies per genome, providing a direct measure of the genetic modification.

Workflow Integration and Logical Relationships

The various assay categories are not used in isolation but are integrated into a cohesive testing strategy to fully characterize a cell therapy product. The following diagram illustrates the logical relationship between these assays and the specific biological or chemical processes they measure.

Figure 1: Logical Workflow of Key Assay Categories in Cell Therapy Potency Testing. This diagram maps the relationship between the four main assay categories and the specific biological attributes they are designed to measure, illustrating how they provide a multi-faceted view of product quality and function.

Research Reagent Solutions

Selecting the appropriate reagents and tools is fundamental to developing robust and reliable potency assays. The following table details key solutions used across the featured assay categories.

Table 2: Essential Research Reagent Solutions for Potency Assays

Item / Solution	Primary Function in Assays	Example Application
Chromium Single Cell Assays	Enables high-throughput single-cell RNA sequencing and multiomic profiling.	Characterizing heterogeneous cell populations and quantifying gene expression at single-cell resolution [29].
Cell Viability Assay Kits	Provides optimized reagents to quantify live and dead cells.	High-throughput screening of compound cytotoxicity in drug discovery [27].
USP Bioassay Reference Standards	Provides a consistent, qualified standard to normalize potency measurements.	Ensuring bioassay results do not drift over time or shift with new reagent lots, critical for release testing and stability studies [28].
Digital PCR Systems	Enables absolute quantification of nucleic acid sequences without a standard curve.	Precisely measuring vector copy number in genetically modified cell therapies [30].
Flow Cytometry Antibodies	Allows for detection and quantification of specific cell surface and intracellular proteins.	Measuring CAR expression levels on engineered T-cells as a key identity and potency assay [8].

A robust potency assay strategy is a cornerstone of successful cell therapy development and regulatory approval. Data from approved products clearly demonstrates that a combination of assays is the standard, with viability and expression tests forming a foundational pair used by the majority of products. The integration of a functional bioassay, while potentially underreported due to redactions, is critical for demonstrating a product's biological effect. The choice and validation of specific assays must be guided by the CTP's unique mechanism of action. By leveraging a matrix of complementary tests—from basic viability and genetic identity to complex functional bioassays—developers can comprehensively ensure the quality, consistency, and potency of their cell therapy products, ultimately supporting their safety and efficacy in the clinic.

In the field of cell-based therapies, a potency assay is a critical quality control test that serves as a surrogate measure of the product's biological function and its intended mechanism of action. For cell therapies designed to promote therapeutic angiogenesis, the quantification of Vascular Endothelial Growth Factor (VEGF) has emerged as a key potency indicator. This case study focuses specifically on the development, validation, and implementation of a VEGF potency assay for ProtheraCytes, an expanded autologous CD34+ cell-based therapy product. The assay's role is to ensure that each clinical batch consistently delivers the intended biological activity—promoting revascularization of damaged myocardial tissue via angiogenesis—before being released for patient administration [31].

The validation of such potency assays must adhere to international guidelines for cell therapy products, establishing criteria for precision, accuracy, linearity, and specificity. This case study will objectively compare the automated VEGF ELISA potency assay for CD34+ cell therapy with other established methods for VEGF bioactivity measurement, providing experimental data and detailed protocols to support the comparative analysis.

The CD34+ Cell Therapy and Its Mechanism of Action

ProtheraCytes CD34+ Cell Therapy

ProtheraCytes are expanded autologous CD34+ cells specifically developed for cardiac repair. The therapeutic premise is that these cells promote regeneration by revascularizing damaged myocardial tissue through angiogenesis. The primary mechanistic driver of this process is the secretion of VEGF by the CD34+ cells, which stimulates the formation of new blood vessels [31]. CD34 is a transmembrane phosphoglycoprotein that serves as a cell-cell adhesion factor and is typically expressed on human hematopoietic progenitor cells and vascular endothelial cells [32].

Research has demonstrated that CD34+/CD90+ adipose-derived stem cells (ASCs) possess significant angiogenic potential, including the capability to form sphere clusters, produce high levels of VEGF, and differentiate into capillary-like structures [33]. These CD34+/CD90+ cells exhibit a high proliferative capacity and can differentiate into endothelial cells expressing CD31, VEGF, and Flk-1 (VEGFR-2) [33].

VEGF Signaling in Angiogenesis

VEGF-A is the most potent angiogenic cytokine and operates through a well-characterized signaling pathway. The binding of VEGF to its primary receptor, VEGFR-2 (KDR), on endothelial cell surfaces initiates receptor dimerization and intracellular signaling cascades. This activation promotes endothelial cell proliferation, migration, and survival—fundamental processes in angiogenesis [34].

The diagram below illustrates the core VEGF-VEGFR2 signaling pathway and the mechanism of its inhibition by neutralizing antibodies, which forms the basis for many potency assays.

The VEGF signaling pathway forms the biological foundation for various bioassays used in potency testing. In reporter gene assays, VEGF binding to VEGFR-2 triggers a signaling cascade that results in the nuclear translocation of the transcription factor NFAT (Nuclear Factor of Activated T-cells). This drives the expression of a luciferase reporter gene, producing a measurable luminescent signal. Anti-VEGF therapeutics, such as bevacizumab and ranibizumab, function by binding to VEGF and preventing this initial receptor interaction, thereby reducing the downstream signal in a concentration-dependent manner [35] [36].

Experimental Protocols for VEGF Potency Assessment

Automated ELISA Potency Assay for CD34+ Cell Therapy

Principle: This potency assay quantifies the level of VEGF secreted by CD34+ cells during their expansion process using an automated ELISA (Enzyme-Linked Immunosorbent Assay) system. The amount of VEGF produced correlates directly with the product's potential to induce therapeutic angiogenesis [31].

Materials and Reagents:

• ProtheraCytes clinical batches (expanded autologous CD34+ cells)
• ELLA automated immunoassay system (ProteinSimple)
• VEGF-specific antibody pairs and reagents
• Cell culture medium
• Validation samples for accuracy, precision, and linearity assessment

Methodology:

• Cell Culture and Supernatant Collection: Expand CD34+ cells following the established clinical manufacturing protocol. Collect culture supernatant at a defined time point post-expansion, which contains the secreted VEGF.
• Automated Immunoassay Execution: Load samples, standards, and controls onto the ELLA system. The system automatically performs all fluidic steps, including sample incubation with VEGF capture antibodies, washing, detection antibody binding, and signal development.
• Quantification: The system measures VEGF concentration in test samples by interpolating from a standard curve. The assay is validated for a linear range of 20 pg/mL to 2800 pg/mL [31].
• Data Analysis: Calculate VEGF concentration for each clinical batch. Compare results against pre-defined specification limits for batch release.

Reporter Gene Assay (RGA) for Anti-VEGF Antibodies

Principle: This cell-based bioassay utilizes HEK293 cells engineered to stably express VEGFR-2 and a luciferase reporter gene under the control of NFAT response elements. VEGF binding to VEGFR-2 activates the signaling cascade, leading to NFAT-mediated luciferase expression. The inhibitory effect of anti-VEGF antibodies is measured as a reduction in luminescence [35] [36].

Materials and Reagents:

• NFAT-RE-Luc2P/KDR HEK293 cells (commercially available from Promega)
• Anti-VEGF therapeutic (bevacizumab, ranibizumab) or control antibodies
• Recombinant human VEGF165
• Luciferase detection reagent
• Cell culture medium and supplements
• White-walled 96-well tissue culture plates
• Luminescence plate reader

Methodology:

• Cell Seeding: Plate cryopreserved, ready-to-use NFAT-RE-Luc2P/KDR HEK293 cells in 96-well plates at a predetermined density.
• Compound Treatment: Pre-incubate a dilution series of the anti-VEGF antibody with a fixed concentration of VEGF165. Add the antibody-VEGF mixture to the cells.
• Incubation and Signal Development: Incubate cells for a defined period (typically 5-6 hours) to allow signaling and reporter gene activation. Add luciferase substrate and measure luminescent signal.
• Data Analysis: Generate a dose-response curve by plotting luminescence (relative light units) against the logarithm of antibody concentration. Calculate the relative potency (IC50) of the test sample compared to a reference standard [35].

Enzyme Fragment Complementation (EFC) Assay

Principle: The PathHunter VEGF Bioassay (Eurofins DiscoverX) uses engineered HEK293 cells expressing VEGFR-2 monomers fused to complementary fragments of β-galactosidase (EA and ED). VEGF-induced receptor dimerization brings these fragments into proximity, restoring enzyme activity, which is measured by chemiluminescence. Anti-VEGF antibodies inhibit this dimerization, reducing the signal [35].

Materials and Reagents:

• PathHunter VEGF Receptor HEK293 Cells (Eurofins DiscoverX)
• Anti-VEGF therapeutics and controls
• Recombinant human VEGF
• PathHunter Detection Reagents
• Cell culture plates and medium
• Luminescence plate reader

Methodology:

• Cell Preparation: Plate the engineered HEK293 cells in growth medium.
• Treatment: Add VEGF pre-incubated with serial dilutions of the anti-VEGF antibody to the cells.
• Incubation: Incubate for a specified period (typically 2-5 hours) to allow receptor activation and dimerization.
• Signal Detection: Add chemiluminescent substrate and measure signal intensity. The generated light signal is proportional to the amount of VEGF-induced receptor dimerization.
• Data Analysis: Plot dose-response curves and calculate IC50 values for potency determination [35].

Comparative Experimental Data and Validation

Validation Data for VEGF Potency Assays

The table below summarizes key validation parameters for three different types of VEGF potency assays, demonstrating their performance characteristics and suitability for various applications.

Table 1: Comparative Validation Data for VEGF Potency Assays

Validation Parameter	CD34+ Cell VEGF ELISA [31]	Reporter Gene Assay [35]	Enzyme Fragment Complementation (EFC) [35]
Assay Time	Not specified	< 8 hours	< 24 hours
Linearity Range	20-2800 pg/mL	50-150% of test concentration	50-150% of test concentration
Linearity (R²)	0.9972	0.9968	0.9899
Precision (Repeatability)	CV ≤ 10%	RSD = 7.0%	Mean RSD = 10.1%
Intermediate Precision	CV ≤ 20%	RSD = 7.6%	Mean RSD = 10.2%
Accuracy (Mean Recovery)	85-105%	85-120%	99.9%
Specificity	Specific for VEGF	Specific for VEGF inhibition; no inhibition with irrelevant antibodies	Not fully evaluated
Key Application	Release testing for CD34+ cell therapy	Potency testing of anti-VEGF antibodies	Potency testing of anti-VEGF antibodies

Performance Comparison of Anti-VEGF Therapeutics

Binding affinity and neutralization potency are critical differentiators among anti-VEGF therapeutics. The following table compiles experimental data comparing these parameters for major anti-VEGF agents under identical experimental conditions.

Table 2: Binding and Neutralization Properties of Anti-VEGF Agents [34]

Anti-VEGF Agent	Type	VEGF-A165 Affinity (K_D)	Relative Potency in Cell-Based Assays	Ligand Binding Specificity
VEGF Trap (Aflibercept)	Soluble decoy receptor	Sub-picomolar	Most potent in inhibiting VEGFR1/VEGFR2 activation, calcium mobilization, and migration	VEGF-A, VEGF-B, PlGF
Ranibizumab	Humanized monoclonal antibody Fab fragment	Lower than VEGF Trap	Less potent than VEGF Trap	VEGF-A only
Bevacizumab	Humanized monoclonal antibody	Lower than VEGF Trap	Less potent than VEGF Trap	VEGF-A only

The significantly higher affinity of VEGF Trap for VEGF-A, coupled with its faster association rate and broader ligand recognition (VEGF-B and PlGF), translates to superior potency in cell-based bioassays compared to antibody-based agents [34].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for VEGF Potency Assays

Reagent / Solution	Function / Application	Examples / Specifications
CD34+ Cells	Therapeutic product for potency testing	ProtheraCytes (expanded autologous CD34+ cells) [31]
Engineered Cell Lines	Bioassay systems for potency testing	NFAT-RE-Luc2P/KDR HEK293 (Promega); PathHunter VEGF Receptor HEK293 (Eurofins DiscoverX) [35]
VEGF Ligands	Stimulating agent in bioassays	Recombinant human VEGF165, VEGF121 isoforms [34]
Anti-VEGF Therapeutics	Reference standards in potency assays	Bevacizumab, Ranibizumab, Aflibercept [34]
Detection Systems	Signal measurement in various assay formats	ELLA automated immunoassay system; Luciferase reagents; PathHunter detection kit [31] [35]
Validation Samples	Assay qualification and validation	Samples for accuracy, precision, linearity, and specificity assessment [31]
Cox-2-IN-21	Cox-2-IN-21, MF:C21H22N6O4, MW:422.4 g/mol	Chemical Reagent
Treprostinil-13C2,d1	Treprostinil-13C2,d1, MF:C23H34O5, MW:393.5 g/mol	Chemical Reagent

Discussion and Comparative Analysis

Assay Selection Considerations

The selection of an appropriate potency assay depends on multiple factors, including the nature of the product (cell therapy vs. biologic drug), regulatory requirements, and practical considerations for implementation in a quality control environment.

The automated VEGF ELISA for CD34+ cell therapy offers significant advantages for its intended application. The validation data demonstrate excellent linearity (R²=0.9972), precision (CV ≤10% for repeatability), and accuracy (85-105% recovery), meeting international guidelines for cell therapy products [31]. Furthermore, its quantitative nature and compatibility with automation make it suitable for the timely release of clinical batches, a critical requirement for autologous cell therapies with limited shelf lives.

Compared to cell-based bioassays, the VEGF ELISA is more straightforward and robust for quantifying a specific secreted factor. However, it measures only one aspect of the product's biological function, whereas cell-based bioassays like the RGA and EFC assays measure the functional consequence of VEGF pathway activation or inhibition.

Advantages and Limitations of Different Platforms

Automated VEGF ELISA:

• Advantages: Highly precise, quantitative, rapid execution, easily automated, well-suited for product release testing.
• Limitations: Measures only VEGF concentration, not biological activity; may not reflect the complete therapeutic mechanism.

Reporter Gene Assay (RGA):

• Advantages: Functional measure of biological activity, high sensitivity (picomolar range), good precision (RSD ~7%), rapid turnaround (<8 hours) [35] [36].
• Limitations: Requires specialized cell line, may have higher variability than biochemical assays.

Enzyme Fragment Complementation (EFC):

• Advantages: Homogeneous assay format (no washing steps), good linearity, specific measurement of receptor dimerization.
• Limitations: Longer assay time (~24 hours), potentially higher variability (RSD ~10%) [35].

Traditional HUVEC Proliferation Assay:

• Advantages: Measures a biologically relevant endpoint (cell proliferation).
• Disadvantages: High variability (due to use of primary cells), lengthy procedure (3-4 days), technically challenging [35] [36].

The following diagram illustrates the fundamental workflow differences between the primary assay types discussed, highlighting their key steps and outputs.

This case study demonstrates that the validated VEGF potency assay for CD34+ cell-based therapy fulfills regulatory requirements for product release while offering robustness, precision, and practical efficiency. The automated ELISA format is particularly suited to the constraints of autologous cell therapy manufacturing, where rapid and reliable quality control is essential for patient-specific batch release.

When compared to alternative potency platforms for VEGF-targeted therapies, each assay system presents distinct advantages and limitations. While cell-based bioassays (RGA, EFC) provide functional assessment of biological activity, they may introduce greater variability and complexity. The traditional HUVEC proliferation assay, despite measuring a biologically relevant endpoint, suffers from high variability and lengthy procedures that make it less suitable for routine quality control [35] [36].

For researchers and drug development professionals selecting potency assay platforms, the decision should be guided by the specific product characteristics, mechanism of action, and intended use of the assay. The successful validation and implementation of the VEGF ELISA for CD34+ cell therapy illustrates how a well-designed potency method can effectively bridge product quality attributes with biological function, ensuring both patient safety and therapeutic efficacy.

The remarkable clinical success of Chimeric Antigen Receptor (CAR) T-cell therapy in treating hematological malignancies has underscored the critical need for robust potency assays that can accurately predict the therapeutic potential of these living drugs [4]. Traditional potency assays have primarily been designed to measure well-established CAR T-cell functionalities, such as cytokine release (e.g., IFN-γ) and immediate cytotoxic activity [4]. However, as CAR T-cell products become increasingly complex and their applications expand into solid tumors, conventional assays may no longer fully capture the multifaceted nature of their biological activity [4] [37].

The emergence of sophisticated multi-omics technologies has revolutionized our understanding of CAR T-cell biology by enabling comprehensive profiling across genomic, epigenomic, transcriptomic, proteomic, and metabolomic dimensions [38]. These advanced approaches have revealed a broader spectrum of critical cellular characteristics that correlate strongly with clinical efficacy, including differentiation states, metabolic fitness, and long-term persistence potential [4] [38]. This paradigm shift necessitates the development of next-generation potency assays that integrate multidimensional data to fully characterize CAR T-cell products and better predict their in vivo performance [4] [38].

Within the broader context of cell therapy potency assay research, this guide objectively compares the performance of multi-omics profiling technologies against conventional assessment methods, providing experimental data and protocols to inform assay selection for CAR T-cell development and quality control.

Comparative Analysis of CAR T-Cell Profiling Technologies

Multi-Omics Technologies for Comprehensive CAR T-Cell Characterization

Table 1: Comparison of Multi-Omics Profiling Technologies for CAR T-Cell Assessment

Omics Approach	Profiling Technology	Key Insights for CAR T-Cell Potency	Regulatory Application
Genomics	DNA sequencing (DNA-seq), TCR sequencing (TCR-seq), Vector integration site analysis	Identifies TCR clonotypic diversity, vector copy number (VCN), γδ T-cell content, and potential genotoxicity risks from integration events [4] [38].	VCN is a mandatory lot-release test for FDA-approved products; integration site analysis is currently informational [4].
Epigenomics	DNA methylation arrays, ATAC-seq, ChIP-seq, scATAC-seq	Defines differentiation states and persistence potential through chromatin accessibility and methylation patterns; identifies epigenetic loci associated with clinical response [4] [38].	Emerging for characterizing cellular states predictive of efficacy; not yet standard for release [4].
Transcriptomics	RNA-seq, scRNA-seq	Reveals gene expression signatures of T-cell subsets, activation, and exhaustion; enables identification of functional states linked to clinical outcomes [4] [38].	Used in research and development to correlate product profiles with potency; scRNA-seq can track clonotype dynamics [4].
Proteomics	Mass spectrometry, Mass cytometry (CyTOF)	Characterizes protein expression, signaling pathways, and surface marker phenotypes; identifies activation and exhaustion markers [38].	Flow cytometry for CAR expression is common in quality control; phosphoproteomics informs signaling strength [38].
Metabolomics	Mass spectrometry, Metabolic flux analysis	Discovers metabolic reprogramming in CAR T-cells; assesses glycolytic activity and mitochondrial fitness linked to persistence [4] [38].	Emerging field with potential to assess metabolic fitness as a quality attribute; not yet standardized for release.

Conventional vs. Advanced Potency Assays

Table 2: Performance Comparison of CAR T-Cell Cytotoxicity and Potency Assays

Assay Method	Measured Parameters	Throughput	Key Advantages	Key Limitations
IFN-γ Release (Traditional)	IFN-γ concentration after antigen exposure [4]	High	Standardized, correlates with activation, used in FDA-approved products [4]	Short-term, single parameter, may not reflect sustained killing [4]
Chromium-51 Release	Specific lysis of labeled target cells [6]	Low	Considered a "gold standard" for cytotoxicity	Radioactive, endpoint measurement only, high variability [6]
Flow Cytometry-Based	Target cell viability (7-AAD), activation markers (CD69), specific cell populations [39] [6]	Medium to High	Multiplexed, provides phenotypic data, high information content [39]	Requires expertise, complex data analysis, semi-quantitative for killing [6]
Impedance-Based (e.g., Maestro Z)	Real-time cell-induced cytolysis via impedance [39]	Medium	Label-free, real-time kinetic data, continuous monitoring [39] [6]	Specialized equipment, measures overall cytolysis without distinguishing effector mechanisms [39]
Bioluminescence-Based (e.g., CLARIOstar)	Residual tumor cell viability via luciferase activity [39]	High	Highly sensitive, quantitative, ideal for ECâ‚…â‚€ determination [39]	Requires genetically modified target cells, endpoint measurement [6]
Long-Term Co-culture (7-day)	Recursive killing, proliferative capacity, cytokine production, phenotype over time [40]	Low	Models high tumor burden, assesses sustained functionality and exhaustion [40]	Labor-intensive, low throughput, longer duration [40]

Experimental Protocols for Key Multi-Omics and Functional Assays

Protocol 1: Single-Cell Multi-Omics Profiling for CAR T-Cell Characterization

Objective: To simultaneously characterize the transcriptomic and epigenomic landscape of CAR T-cell products at single-cell resolution, enabling the identification of cellular states predictive of potency [4] [38].

Materials:

• CAR T-cell product (cryopreserved or fresh)
• Single-cell multi-ome ATAC + Gene Expression kit (10x Genomics)
• Chromium Controller (10x Genomics)
• Next-generation sequencer (Illumina recommended)
• Cell Ranger ATAC and Cell Ranger ARC analysis pipelines
• R or Python environment with Seurat, ArchR, or similar packages

Methodology:

• Cell Viability and Preparation: Thaw CAR T-cells if cryopreserved and assess viability using trypan blue exclusion. Ensure viability >80%. Wash and resuspend cells in PBS + 0.04% BSA at a concentration of 1,000-1,200 cells/μL.
• Library Preparation: Load the cell suspension onto the Chromium chip to target recovery of 5,000-10,000 cells. Proceed with tagmentation and GEM generation according to the manufacturer's protocol. This simultaneously captures chromatin accessibility and mRNA from the same cell.
• Sequencing: Construct libraries and sequence on an Illumina platform. Aim for >20,000 read pairs per nucleus for ATAC data and >50,000 read pairs per cell for gene expression.
• Data Analysis:
  • Use Cell Ranger ARC to demultiplex data and align reads to the reference genome.
  • Perform quality control: remove cells with low unique nuclear fragments (<1,000) or high mitochondrial read percentage (>20%).
  • Integrate ATAC and gene expression data to define distinct CAR T-cell clusters.
  • Annotate clusters using canonical markers (e.g., SELL for naive, CCR7 for central memory, GZMB for effector).
  • Identify differentially accessible chromatin regions and correlated gene expression linked to functional states.

Supporting Data: A study applying paired single-cell RNA and TCR repertoire profiling to 24 infusion products found that products associated with poor clinical responses exhibited reduced TCR clonotypic diversity and showed transcriptional exhaustion signatures [4].

Protocol 2: Long-Term Co-culture Potency Assay for Recursive Killing

Objective: To evaluate the long-term cytotoxic function and proliferative capacity of CAR T-cells under conditions of high tumor burden, which better mimics physiological challenges than short-term assays [40].

Materials:

• CAR T-cell effector cells
• Target tumor cells (e.g., Raji-luc for CD19-CAR)
• Cell culture medium (e.g., RPMI-1640 + 10% FBS)
• Luminescent cell viability assay reagent (e.g., RealTime-Glo MT Cell Viability Assay, or luciferin if using luciferase-expressing targets)
• Plate reader capable of luminescence detection
• Flow cytometer for phenotypic analysis

Methodology:

• Initial Co-culture Setup: Seed target cells (e.g., 50,000 cells/well) in a 24-well plate. Add CAR T-cells at the desired effector-to-target (E:T) ratio (e.g., 1:1). Include wells with target cells alone as a control.
• Long-Term Co-culture: Culture cells for 7 days at 37°C, 5% COâ‚‚. Do not replenish medium during this period to simulate a high-burden, nutrient-restricted environment.
• Assessment of Recursive Killing:
  • Day 3 and Day 7: Gently resuspend cells and remove an aliquot for viability assessment.
  • For luminescent assays, add substrate and measure signal. Calculate specific lysis: [1 - (Luminescence E:T / Luminescence Target Alone)] × 100.
  • Alternatively, count viable cells using trypan blue exclusion.
• Concurrent Phenotypic and Functional Analysis:
  • At endpoints, analyze CAR T-cell expansion by counting absolute numbers.
  • Use flow cytometry to profile for exhaustion markers (e.g., PD-1, TIM-3, LAG-3).
  • Collect supernatant for multiplex cytokine analysis (IFN-γ, TNF-α, IL-2).

Supporting Data: This method provides a facile approach to elucidate functional variations across different CAR T-cell products, assessing their proliferative capacity and sustained cytotoxic function critical for in vivo efficacy [40].

Visualization of Multi-Omics Integration in CAR T-Cell Profiling

Multi-Omics Integration for Potency Assessment

The Scientist's Toolkit: Essential Reagents and Research Solutions

Table 3: Key Research Reagent Solutions for CAR T-Cell Potency Assays

Reagent / Tool	Function	Application in Potency Assessment
Xynapse-T Reagents	Synthetic antigen-presenting molecules (pMHC + CD28 engagement) [41]	Replaces variable biological APCs for standardized T-cell activation assays; measures cytokine production and activation in a highly reproducible system [41].
Single-Cell Multi-Ome Kit	Simultaneously profiles gene expression and chromatin accessibility from single cells [4] [38]	Defines CAR T-cell functional states and epigenetic heterogeneity within products, correlating subsets with potency.
RealTime-Glo MT Cell Viability Assay	Non-lytic, luminescent assay for real-time monitoring of cell viability [39] [6]	Enables kinetic assessment of CAR T-cell-mediated cytotoxicity over time in long-term co-culture assays.
Magnetic Cell Separation Beads	Isolation of specific T-cell subsets (e.g., CD4+, CD8+, Naive T cells) [4]	Allows for manufacturing of defined CAR T-cell products with optimized composition, impacting potency and persistence.
Luminescent Caspase Assays	Measures caspase activity as an early marker of apoptosis in target cells [6]	Provides a sensitive, homogeneous method to quantify cytotoxic activity in a high-throughput format.
Multiplex Cytokine Panels	Quantifies multiple cytokines (IFN-γ, TNF-α, IL-2, etc.) from a single sample [4] [40]	Profiles the polyfunctional capacity of CAR T-cells, which can be a critical marker of potency.
BPR1R024 mesylate	BPR1R024 mesylate, MF:C25H25F3N6O5S, MW:578.6 g/mol	Chemical Reagent
Bromo-PEG4-PFP ester	Bromo-PEG4-PFP ester, MF:C17H20BrF5O6, MW:495.2 g/mol	Chemical Reagent

The integration of multi-omics approaches with conventional potency assays represents the future of CAR T-cell product characterization. While traditional methods like IFN-γ release remain important for lot-release testing, they provide an incomplete picture of the complex biological attributes that determine clinical success [4]. Genomic, epigenomic, transcriptomic, proteomic, and metabolomic profiling collectively offer a powerful, multidimensional view of CAR T-cell potency, revealing critical insights into persistence, differentiation states, metabolic fitness, and functional heterogeneity [4] [38].

For researchers and drug development professionals, the strategic implementation of these advanced profiling technologies—particularly in early product development and characterization—is essential for designing more effective and predictable CAR T-cell therapies. As the field progresses towards increasingly complex products for solid tumors and other challenging indications, potency assays will likely evolve to incorporate specific multi-omics signatures that correlate strongly with clinical outcomes, ultimately ensuring that patients receive cell therapies with predictable and potent therapeutic activity.

Optimizing Potency Assay Strategy to Accelerate Development and Avoid Pitfalls

In the rapidly advancing field of cell and gene therapy, potency assays have traditionally been viewed as a final checkpoint before product release—a regulatory requirement to be addressed late in the development pipeline. However, this perspective is fundamentally changing as developers embrace an accelerator mindset that integrates critical potency assessment from the earliest research stages. This paradigm shift recognizes that comprehensive product characterization beginning in discovery phases significantly de-risks development and accelerates regulatory approval [4]. The growing complexity of "living drugs," including CAR T-cells, TCR-engineered products, and Tumor-Infiltrating Lymphocytes (TILs), demands a more sophisticated approach to potency evaluation that captures their multifaceted biological activity [42].

Potency, defined as the quantitative measure of biological activity specific to the product's mechanism of action (MoA), serves as a critical quality attribute (CQA) that must be monitored throughout development [43]. For cell therapies, this extends beyond simple cytotoxicity measurements to encompass viability, expansion capacity, persistence, and differentiation states [4]. This guide examines current and emerging potency testing strategies, providing objective comparisons and experimental data to help researchers select appropriate assays for their specific development context. By adopting this integrated approach, developers can establish a robust potency assessment framework that enhances product understanding, ensures consistency, and ultimately brings effective therapies to patients more efficiently.

Comparative Analysis of Cell-Based Potency Assay Platforms

Cytotoxicity Assays: Technical Comparison

Cytotoxicity represents a fundamental MoA for most cell therapies, particularly CAR T-cell products. Table 1 provides a technical comparison of four commonly used cytotoxicity assay platforms, highlighting their respective advantages, limitations, and optimal use cases [44].

Table 1: Comparison of Cell-Mediated Cytotoxicity Assay Platforms

Assay Format	Chromium Release	Bioluminescence	Impedance	Flow Cytometry
Principal Measure	51Cr release	Luciferase activity	Cell detachment	Live/dead staining, phenotype
Radioactive Materials	Yes	No	No	No
Target Cell Labeling	Yes	No	No	Yes
Genetic Target Modification	No	Yes (reporter gene)	No	No
Measurement Type	Endpoint	Endpoint	Temporal	Endpoint
Real-time Measurement	No	No	Yes	No
Maximum Time Point	18–24 hours	Days	Days	Days
Heterogenous Target Assessment	No	No	No	Yes
Throughput & Automatability	Low	High	High	High

The impedance-based and bioluminescence assays offer significant advantages for early development due to their kinetic measurement capabilities and higher throughput, allowing researchers to monitor dynamic cell-killing profiles over time rather than at a single endpoint [44]. The flow cytometry platform provides unique value in assessing differential cytotoxicity on heterogenous target cell populations, which better mimics the physiological tumor microenvironment [44].

Advanced Functional Assays: Predicting Potency and Persistence

While cytotoxicity remains essential, a comprehensive potency assessment must evaluate additional critical functionalities. Recent advances include assays that model the tumor microenvironment (TME) to better predict in vivo performance [16]. One innovative approach manufactures CAR T-cells under hypoxic and hyperbaric conditions, which induces an increased stem-like phenotype and enhances long-term serial killing activity—key determinants of clinical persistence [16].

The AVATAR platform represents another technological advancement that enables simultaneous assessment of cytokine secretion profiles (IFN-γ, TNF-α, IL-2) alongside direct functional measurements of target cell viability [16]. This integrated approach addresses the limitation of cytokine-only assays, which lack direct information on target cell status and long-term persistence [16].

Multi-Omics Approaches in Potency Profiling

Comprehensive Molecular Characterization

The accelerator mindset emphasizes deep product characterization using multi-omics approaches throughout development. Table 2 outlines key profiling methodologies that provide complementary insights into product potency [4].

Table 2: Multi-Omics Profiling Approaches for CAR T-Cell Products

Profiling Method	Key Parameters	Potency Relevance	Technology Examples
Genomic	Vector copy number (VCN), Integration sites, TCR repertoire	Safety, Clonal expansion, Persistence	ddPCR, INSPIIRED pipeline, EpiVIA, TCR-seq [4]
Epigenomic	DNA methylation, Chromatin accessibility, Histone modifications	Differentiation state, Exhaustion, Memory potential	Bulk and single-cell ATAC-seq, Methylation arrays [4]
Transcriptomic	Gene expression patterns, T-cell subsets	Functional states, Activation, Exhaustion	Bulk and single-cell RNA-seq [4]
Proteomic	Surface/Intracellular protein expression, Signaling	Activation status, Effector functions, Persistence	Flow cytometry, Mass cytometry [4]
Metabolomic	Energy metabolism, Metabolic intermediates, Enzymes	Metabolic fitness, Proliferation capacity	Glycolytic activity, Mitochondrial fitness assays [4]

Experimental Workflow for Multi-Omics Potency Assessment

Implementing these multi-omics technologies requires a structured workflow that aligns with development phases. The following diagram illustrates an integrated approach to potency assessment:

Integrated Potency Assessment Workflow

This workflow emphasizes parallel molecular and functional characterization, with data integration enabling identification of potency signatures that correlate with clinical outcomes [4]. For example, epigenomic profiling of DNA methylation patterns in CD19 CAR T-cell products has identified 18 distinct epigenetic loci associated with complete response, event-free survival, and overall survival [4].

Essential Research Reagent Solutions

Implementing robust potency assays requires specific reagent systems and tools. Table 3 details key solutions that support comprehensive potency assessment throughout development.

Table 3: Essential Research Reagent Solutions for Potency Assay Development

Reagent Category	Specific Examples	Function in Potency Assessment	Application Context
Reporter Cell Lines	iLite technology with chimeric transcription factors [45]	Measures biological activity via reporter gene expression; includes normalization readout	Potency testing for biologics and biosimilars
Cell Viability/Tracking Dyes	CFSE, CellTrace Violet, CMTMR, DiO, 7-AAD, Propidium Iodide [44]	Labels target/effector cells for tracking; distinguishes live/dead populations	Flow cytometry-based cytotoxicity assays
Cytokine Detection Assays	IFN-γ, TNF-α, IL-2 secretion assays [16]	Quantifies cytokine release as measure of T-cell activation	Functional potency assessment
Phenotypic Characterization Antibodies	CD3, CD4, CD8, CD45RA, CD62L, CD197 [4]	Identifies T-cell differentiation subsets (naïve, memory, effector)	Product characterization and potency correlation
TCR Repertoire Analysis Kits	Bulk and single-cell TCR sequencing kits [4]	Assesses TCR clonotypic diversity as surrogate for persistence	Genomic profiling of infusion products
Metabolic Assay Kits	Glycolytic rate, Mitochondrial stress test kits [4]	Evaluates metabolic fitness and energy pathways	Metabolomic profiling for persistence prediction

These reagent solutions enable researchers to implement the assay platforms discussed in previous sections. For example, cell tracking dyes like CFSE and CellTrace Violet are essential for flow cytometry-based cytotoxicity assays, allowing differential labeling of target and effector cells [44]. Similarly, TCR repertoire analysis tools provide critical insights into the clonal composition of therapeutic products, which has been correlated with clinical responses [4].

Detailed Experimental Protocols

Protocol 1: Flow Cytometry-Based Cytotoxicity Assay

This protocol provides a method for quantifying cell-mediated cytotoxicity, adapted for high-throughput screening during early development [44].

Materials:

• Effector cells (CAR T-cells or other cell therapy product)
• Target cells (appropriate for MoA)
• Cell staining buffers (PBS + 2% FBS)
• CFSE or CellTrace Violet cell proliferation dyes
• 7-AAD or propidium iodide viability dyes
• Flow cytometer with plate reader capability

Procedure:

• Target Cell Labeling:
  • Harvest and wash target cells in PBS
  • Resuspend at 1-2×10^6 cells/mL in pre-warmed PBS containing 0.5-1μM CFSE or CellTrace Violet
  • Incubate for 20 minutes at 37°C protected from light
  • Quench staining with 5 volumes of complete media and incubate for 5 minutes
  • Wash cells twice with complete media and resuspend at appropriate concentration

• Effector Cell Preparation:

  • Harvest and wash effector cells
  • Count and resuspend in complete media at highest effector:target (E:T) ratio
  • Prepare serial dilutions for multiple E:T ratios

• Coculture Setup:

  • Plate 5,000-20,000 labeled target cells per well in 96-well U-bottom plates
  • Add effector cells at predetermined E:T ratios (typically 50:1 to 1:1)
  • Include target cells alone (spontaneous death control) and with lysis buffer (maximum death control)
  • Centrifuge plates briefly (500×g for 2 minutes) to initiate cell contact
  • Incubate for 4-24 hours at 37°C, 5% CO2 based on expected kinetics

• Sample Processing and Analysis:

  • Transfer cells to V-bottom plates and wash with staining buffer
  • Add viability dye (7-AAD or propidium iodide) 5-10 minutes before acquisition
  • Acquire data on flow cytometer, collecting minimum of 1,000 target cell events
  • Analyze target cell death by quantifying viability dye-positive population within labeled target cell gate

• Data Analysis:

  • Calculate specific lysis using formula: % Specific Lysis = (Experimental % Dead - Spontaneous % Dead) / (Maximum % Dead - Spontaneous % Dead) × 100
  • Generate dose-response curves across E:T ratios
  • Determine area under curve (AUC) for potency comparisons between products

Protocol 2: Multi-Omics Potency Signature Profiling

This advanced protocol enables comprehensive molecular characterization of cell therapy products to establish correlations with functional potency [4].

Materials:

• High-quality CAR T-cell product (≥1×10^6 cells)
• RNA/DNA extraction kits with quality assessment capability
• Single-cell RNA sequencing kit (10x Genomics or equivalent)
• ATAC sequencing kit
• TCR sequencing kit
• Methylation array or sequencing platform
• Droplet digital PCR (ddPCR) system for VCN quantification

Procedure:

• Sample Preparation for Multi-Omic Analysis:
  • Aliquot cell product for parallel molecular and functional analyses
  • Preserve 1×10^6 cells in RNA/DNA stabilization buffer for bulk analyses
  • Prepare single-cell suspensions for 10x Genomics profiling (200-500 viable cells/μL)
  • Flash-freeze cell pellets for metabolomic analyses where applicable

• Genomic Analyses:

  • Extract genomic DNA and quantify using spectrophotometry/fluorometry
  • Perform ddPCR for vector copy number (VCN) quantification
  • Conduct TCR sequencing to assess repertoire diversity
  • Analyze integration sites using INSPIIRED or EpiVIA pipelines for research applications

• Epigenomic Profiling:

  • Perform ATAC sequencing to assess chromatin accessibility
  • Conduct methylation profiling using array or sequencing approaches
  • Identify differentially accessible regions between high- and low-potency products

• Transcriptomic Analysis:

  • Extract high-quality RNA (RIN ≥8.0)
  • Perform bulk RNA sequencing for overall transcriptional profiling
  • Conduct single-cell RNA sequencing to resolve cellular heterogeneity
  • Identify gene signatures associated with favorable differentiation states

• Data Integration and Correlation:

  • Align molecular data with functional potency measurements
  • Identify consensus signatures predictive of product performance
  • Develop simplified scoring algorithms for routine potency assessment

Regulatory Considerations and Future Directions

The regulatory landscape for cell therapy potency testing continues to evolve as products increase in complexity. Regulatory agencies emphasize the importance of phase-appropriate assay validation and mechanistically relevant potency measures [43]. As highlighted in recent guidelines, potency tests should be validated to ICH Q2(R2) standards, with assays specifically tailored to the product's MoA [43].

Emerging trends in potency testing include the development of complex tumor microenvironment models that better predict in vivo performance, and the application of artificial intelligence to predict product success based on multi-omics signatures [42]. Some research groups are working toward a "ChatGPT for engineered immune cells"—a program that can predict the most logical next steps for successful cell designs without extensive experimentation [42]. These advances, coupled with the infrastructure to rapidly translate discoveries from laboratory to clinic through GMP simulator environments, position the field to more effectively bring next-generation cell and gene therapies to patients [42].

By adopting an accelerator mindset and integrating comprehensive potency assessment throughout development, researchers can de-risk their programs, accelerate regulatory approval, and ultimately deliver more effective cellular therapies to patients.

The development of robust potency assays is a fundamental requirement for the successful clinical translation and regulatory approval of cell therapies. Potency, defined as the quantitative measure of a product's biological activity, must be linked to relevant biological properties and ideally to the product's clinical mechanism of action [2]. For Advanced Therapy Medicinal Products (ATMPs), which include cell and gene therapies, potency testing presents unique challenges due to inherent biological variability, complex mechanisms of action, and the living nature of these products [46]. The absence of standardized reference materials and consistent assay methodologies across the industry creates significant barriers to product comparability, manufacturing consistency, and reliable potency assessment. An analysis of the 31 US FDA-approved cell therapy products reveals that these products employ an average of 3.4 potency tests each, with the most common being viability/count measurements (52%) and expression markers (27%), while only 23% publicly report bioassays due to extensive redactions of proprietary information [9] [8]. This article examines the current landscape of cell therapy potency testing, identifies key barriers to standardization, and provides comparative experimental data and methodologies to advance the field toward greater consistency and reliability.

Current Landscape of Cell Therapy Potency Testing

Regulatory Framework and Requirements

Global regulatory agencies, including the FDA and EMA, mandate that cell therapy products must include potency testing as part of lot-release testing to ensure product quality, manufacturing consistency, and stability [9] [46]. The regulatory expectation is that potency assays should quantitatively measure biological activities linked to the product's mechanism of action, with the goal of assuring that the product can achieve its intended biological effect [2]. However, the field faces a significant challenge in developing appropriate potency tests that fully capture complex product functionalities while meeting regulatory standards. The development of potency assays typically begins during preclinical and early clinical development, with validation expected prior to Phase III clinical trials [2]. A comprehensive analysis of FDA-approved products shows that 84% of cell therapies cite physicochemical assays as potency tests, while the prevalence of bioassays remains unclear due to redactions of proprietary information in regulatory documentation [8].

Analysis of Potency Tests in Approved Products

Table 1: Distribution of Potency Test Types Across 31 FDA-Approved Cell Therapy Products

Potency Test Category	Number of Tests	Percentage of Total	Example CTPs Utilizing This Test
Viability and Count	37	52%	Hemacord, Clinimmune, Ducord
Expression	19	27%	Kymriah, Yescarta, Tecartus
Genetic Modification	6	9%	Zynteglo, Skysona
Bioassays	7	7%	Abecma, Tecelra
Histology	2	3%	Gintuit, Rethymic
Redacted/Proprietary	33	32%	Breyanzi, Aucatzyl

Analysis of FDA-approved cell therapy products reveals distinctive patterns in potency testing strategies across product categories [8]. Hematopoietic stem cell-cord blood products utilize the highest number of potency tests per product (average 4.4), reflecting their complex biological composition. In contrast, CAR T-cell products employ relatively few potency tests (average 1.9), typically combining viability assessment, CAR expression measurement, and in some cases functional bioassays such as IFN-γ release upon target cell stimulation [9]. Tissue-engineered products demonstrate the most varied approaches, employing histology-based assessments that evaluate structural characteristics like epidermal coverage, dermal thickness, and matrix integrity [9]. This diversity in testing approaches highlights the product-specific nature of potency assessment and the challenge of establishing standardized methods across different therapeutic modalities.

Critical Barriers to Standardization and Consistency

Product Complexity and Biological Variability

Cell therapies present unique standardization challenges due to their inherent biological complexity and manufacturing variability. Unlike traditional pharmaceuticals with well-defined chemical structures, cell therapies comprise living entities with heterogeneous characteristics that can be influenced by donor genetic factors, epigenetic differences, and transcriptional regulation diversities [2]. This biological variability is compounded by complex manufacturing processes involving multiple steps such as cell mobilization, collection, subset isolation, in vitro stimulation, and culture expansion [2]. The limited stability of final cellular products further complicates potency testing, as assays must often be completed within short timeframes between production completion and patient administration [2]. Additionally, the patient-specific nature of autologous therapies means that each manufacturing lot is unique, creating significant challenges for traditional standardization approaches that rely on consistent reference materials across product batches.

Limitations in Current Potency Assay Methodologies

Traditional potency assays for cell therapies often fail to capture the full spectrum of biological activities relevant to clinical efficacy. For CAR T-cell products, conventional potency assessments typically measure IFN-γ release in response to target cells, cell viability, and CAR expression levels [4] [9]. However, emerging research indicates that these assays may not fully reflect critical product characteristics such as differentiation states, metabolic fitness, and long-term persistence potential [4]. The integration of multi-omics approaches—including genomics, epigenomics, transcriptomics, proteomics, and metabolomics—has revealed a broader spectrum of CAR T-cell characteristics that correlate with function but are not captured by conventional potency assays [4]. This gap between conventional testing and product complexity is further widened by the limited availability of standardized reference materials qualified for use across different assay platforms and laboratory environments.

Comparative Analysis of Mycoplasma Testing Methodologies

Experimental Protocol for Mycoplasma Detection

Mycoplasma testing represents a critical safety requirement for cell therapy products, with regulatory standards requiring sensitive detection of contamination. The following experimental protocol was adapted from a comprehensive comparison of five commercial molecular Mycoplasma detection assays [47]:

• Organism Preparation: Ten cultured Mollicutes species were selected based on pharmacopeia standards and reported cell culture contaminants, including Acholeplasma laidlawii, Mycoplasma arginini, M. fermentans, M. gallisepticum, M. hominis, M. hyorhinis, M. orale, M. pneumoniae, M. pulmonis, and Spiroplasma ixodetis.

• Culture Conditions: Organisms were cultured in Hayflick's broth and agar or SP4 medium with arginine as needed, with incubation at 37°C (30°C for S. ixodetis) until metabolic growth was indicated by color change or colonies were visible under dissection scope.

• Sample Preparation: Serial log-fold dilutions were prepared for each organism (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n=6).

• Inhibition Testing: Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs) to evaluate matrix interference.

• Data Analysis: Limit of detection (LOD) was determined for each platform, with repeatability assessed across replicates by two different analysts.

Performance Comparison of Commercial Assays

Table 2: Performance Characteristics of Commercial Mycoplasma Detection Assays

Assay Name	Manufacturer	LOD (CFU/mL)	Meeting ≤10 CFU/mL Standard	Interference with TILs	Key Strengths
Biofire Mycoplasma	bioMérieux Industry	Most sensitive	Yes	No	Highest sensitivity
MycoSEQ	Life Technologies	Comparable to MycoTOOL	Yes	No	Established platform
MycoTOOL	Roche	Comparable to MycoSEQ	Yes	No	Harmonized with standards
VenorGEM qOneStep	Minerva Biolabs	Variable	No for some organisms	No	Rapid workflow
ATCC Universal	American Type Culture Collection	Least sensitive	No	No	Broad organism panel

The comparative analysis revealed significant differences in the performance of commercially available Mycoplasma detection assays [47]. The Biofire Mycoplasma assay demonstrated the highest sensitivity, followed by comparable performance between the MycoSEQ and MycoTOOL assays. Importantly, not all commercial assays met the ≤10 CFU/mL LOD standard required to replace culture-based methods according to European and Japanese pharmacopeia standards. None of the assays showed interference when testing in the presence of tumor infiltrating lymphocytes, indicating their suitability for cellular therapy products without matrix inhibition concerns [47]. This comparative data provides valuable guidance for selecting appropriate safety testing methodologies based on sensitivity requirements and regulatory compliance needs.

Emerging Solutions and Standardization Approaches

Advanced Molecular Methodologies

The development of robust quantitative PCR (qPCR) and digital PCR (dPCR) assays represents a significant advancement in standardized potency testing for cell and gene therapies [48]. These molecular methodologies provide precise quantification of critical quality attributes such as vector copy number (VCN), transgene expression, and cellular persistence. The following experimental protocol outlines best practices for PCR assay validation:

• Primer and Probe Design: Design at least 3 primer-probe sets using specialized software (e.g., PrimerQuest, Primer Express) with customization of PCR parameters. Empirically test candidates in relevant biological matrices and confirm specificity against host genome/transcriptome.

• Assay Validation Parameters: Establish linearity, accuracy, precision, specificity, and robustness following cross-industry recommendations. Include acceptance criteria for PCR efficiency (90-110%), R² >0.98, and inter-assay precision <30% CV.

• Platform Selection: Utilize dPCR for absolute quantification without standard curves, particularly suitable for complex matrices. Employ qPCR for high-throughput applications where relative quantification suffices.

• Context-Based Application: Tailor validation approach based on context of use—biodistribution, transgene expression, viral shedding, or cellular kinetics [48].

The emergence of industry-wide consensus recommendations for PCR assay validations facilitates a more harmonized approach to molecular characterization of cell therapies, addressing the previous lack of regulatory guidance specific to these novel modalities [48].

Multi-Omics Profiling for Comprehensive Potency Assessment

Advanced multi-omics approaches are revolutionizing potency assessment by enabling comprehensive characterization of cell therapy products at multiple molecular levels [4]. The following dot language diagram illustrates how these technologies can be integrated into a standardized potency assessment framework:

This integrated approach enables correlation of molecular characteristics with product functionality, moving beyond conventional single-parameter potency tests. For example, genomic profiling assesses vector copy number and TCR repertoire diversity; epigenomic analysis evaluates DNA methylation patterns associated with T-cell differentiation states; transcriptomics characterizes gene expression signatures of functional subsets; proteomics quantifies CAR expression levels and activation markers; and metabolomics profiles energy pathways indicative of metabolic fitness [4]. By establishing standardized panels of omics-based assays, the field can develop more predictive potency assessment frameworks that better reflect product complexity and clinical performance.

Essential Research Reagent Solutions

Table 3: Key Research Reagents for Cell Therapy Potency Testing

Reagent Category	Specific Examples	Function in Potency Testing	Quality Requirements
PCR Reagents	Primer-probe sets, dPCR mastermix, qPCR reagents	Quantification of VCN, transgene expression, cellular kinetics	GMP-grade if for lot release, validated specificity and sensitivity
Flow Cytometry Reagents	CAR detection antibodies, viability dyes, subset markers	Measurement of CAR expression, viability, immunophenotype	Validated specificity, minimal lot-to-lot variability
Cell Culture Reagents	Target cell lines, cytokine standards, culture media	Functional bioassays, expansion potential	Standardized reference materials, documented stability
Mycoplasma Detection	Biofire Mycoplasma assay, MycoSEQ, MycoTOOL	Sterility testing for product release	Meeting ≤10 CFU/mL sensitivity, no matrix interference
Omics Reagents	Single-cell RNA-seq kits, ATAC-seq reagents, antibody panels	Multi-omics characterization of product attributes	Batch-to-batch consistency, comprehensive validation

The selection of appropriate research reagents is critical for robust potency testing, with quality documentation, lot-to-lot consistency, and performance validation being essential considerations [47] [48]. For regulatory-compliant testing, reagents should be appropriately qualified for their intended use, with particular attention to specificity, sensitivity, and reproducibility. The emergence of standardized reagent panels for multi-omics applications enables more consistent characterization across different products and manufacturing platforms, facilitating comparability assessments and technical transfer between laboratories [4].

The development of standardized reference materials and consistent assay methodologies remains a critical challenge in the field of cell therapy potency testing. Current approaches across FDA-approved products demonstrate considerable diversity, reflecting the product-specific nature of potency assessment and the absence of harmonized standards. The integration of advanced molecular methodologies, comprehensive multi-omics profiling, and standardized reagent panels provides a pathway toward more consistent and predictive potency assessment. The comparative experimental data presented in this analysis offers actionable insights for selecting and validating testing methodologies based on performance characteristics and regulatory requirements. As the field continues to evolve, collaborative efforts between industry stakeholders, regulatory agencies, and standards organizations will be essential to establish robust frameworks for potency testing that ensure product quality while accommodating the inherent biological complexity of cellular therapies.

The development of robust, clinically predictive potency assays remains a formidable scientific and regulatory hurdle for cell therapy developers. An analysis of regulatory filings reveals that potency-related issues are a leading cause of delays and setbacks in the approval pathway for Advanced Therapy Medicinal Products (ATMPs) [20]. This guide examines high-profile case studies where inadequate potency testing strategies stalled product advancement, providing comparative experimental data and actionable protocols to help researchers design assays that withstand regulatory scrutiny.

The Regulatory Imperative: Why Potency Assays Fail

Potency is defined as the quantitative measure of a biological product's specific ability to effect a given result [19]. For cell therapies, a potency assay must reflect the product's intended mechanism of action (MoA) and ideally correlate with clinical response [19] [49]. Regulatory delays occur when sponsors cannot demonstrate this link through a validated, quantitative method.

The scale of the problem is significant: nearly 50% of ATMP applications reviewed by the European Medicines Agency encounter major potency-related issues, and former FDA CBER Director Peter Marks has noted that potency problems are among the most common reasons for delayed approvals in cell and gene therapy [20].

Quantitative Impact of Potency-Assay Delays

The consequences of these delays extend beyond regulatory timelines to affect both patients and developers.

Table 1: Impact of Potency Testing Delays on Cell Therapy Development

Impact Area	Quantitative Effect	Source
Patient Access	20-25% of patients may lose performance status/month without effective therapy for aggressive cancers [20]	NCCN Guidelines, 2024
Cumulative Patient-Days Lost	3,000 patient-days (8+ cumulative years) lost per 100-patient batch delayed by one month [20]	BioBridge Global Analysis
Financial Impact	$5-15 million in lost revenue per month of delay for a high-value therapy [20]	Precedence Research, 2024
Regulatory Success Rate	Major potency issues noted in almost 50% of all ATMP Marketing Authorization Applications in the EU [19]	Barkholt et al.

Case Study Analysis: Regulatory Setbacks and Solutions

Case Study 1: Lifileucel (Autologous Tumor Infiltrating Lymphocyte Therapy)

The development journey of lifileucel, an autologous tumor infiltrating lymphocyte (TIL) therapy, exemplifies the regulatory challenges of demonstrating potency for complex cell products.

• Initial Regulatory Setback: In 2021, the sponsor's original testing scheme, which relied on a single potency assay, was deemed inadequate by the FDA and rejected [50]. The agency determined that a single measurement could not sufficiently capture the multifaceted biological activity of the TIL product.
• Root Cause Analysis: The fundamental issue was a mismatch between the product's complex, multi-mechanistic MoA and an oversimplified analytical approach. TIL therapies function through multiple mechanisms including tumor cell recognition, cytokine release, and direct cytotoxicity—none of which could be fully represented by a single assay.
• Resolution Strategy: The sponsor developed and implemented a comprehensive test matrix incorporating multiple complementary assays. This included a functional cell co-culture assay specifically designed to measure several aspects of the product's biological activity simultaneously [50]. This matrix approach successfully addressed regulatory concerns by providing a more complete characterization of the product's functional potency.

Case Study 2: Mesoblast's Remestemcel-L

While less detailed in publicly available documents, the FDA Advisory Committee Review of Mesoblast's remestemcel-L in 2020 highlighted significant concerns regarding the product's potency assays [8]. The core issue appeared to be insufficient demonstration that the chosen assays adequately measured the critical biological functions responsible for the purported clinical effects.

The Assay Matrix Solution: Learning from Approved Therapies

Analysis of the 31 US FDA-approved cell therapy products (CTPs) reveals that successful regulatory strategies employ multiple complementary potency tests rather than relying on a single method.

Table 2: Potency Test Distribution Across 31 FDA-Approved Cell Therapy Products

Potency Test Category	Frequency of Use	Representative Examples
Viability and Count	37 tests (52%)	Cell viability, total nucleated cell count [8]
Expression	19 tests (27%)	CAR expression from viable T cells, surface marker expression [8]
Bioassays	7 tests (7%)	Cytokine release, cytotoxicity assays [8]
Genetic Modification	6 tests (9%)	Vector copy number, transduction efficiency [8]
Histology	2 tests (3%)	Tissue morphology assessment [8]

This data demonstrates that regulatory success typically requires a multi-attribute approach, with each CTP employing an average of 3.4 distinct potency tests [8]. The most common combination pairs "Viability and Count" with "Expression" measurements, occurring in 16 of the 31 approved CTPs (52%) [8].

CAR-T Cell Potency Assay Matrix

For genetically modified products like CAR-T cells, the regulatory expectation is particularly rigorous. The following workflow illustrates the comprehensive multi-omics approach required for thorough potency assessment:

Diagram 1: Comprehensive CAR-T Cell Potency Assessment Workflow. This integrated approach combines multi-omics characterization with functional assays to fully capture product complexity and predict clinical performance [4].

Experimental Protocols: Developing Robust Potency Assays

Protocol: Cell-Based Relative Potency Assay for AAV Vectors

The validated potency assay for AAV2-hRPE65v2 (Luxturna) provides a template for developing quantitative, mechanism-based potency assays for gene therapies [7].

• Principle: Measures the isomerohydrolase activity of RPE65 by quantifying conversion of all-trans-retinol to 11-cis-retinol via LC-MS/MS.
• Cell Line: HEK293 cells genetically modified to express lecithin retinol acyltransferase (LRAT).
• Procedure:
  • Transduce HEK293-LRAT cells with AAV2-hRPE65v2 at nine different multiplicities of infection (MOIs).
  • Prepare cell lysates and quantify protein concentration.
  • Perform enzymatic assay using nonradioactive all-trans-retinol and CRALBP.
  • Detect and quantify 11-cis-retinol product by LC-MS/MS.
  • Analyze relative potency using a three-parameter logistic (3PL) model and parallel line analysis.

Table 3: Validation Parameters for AAV2-hRPE65v2 Potency Assay

Validation Parameter	Acceptance Criteria	Experimental Results
System Suitability	90% CI for relative potency within 76%-130%	All assays met criteria [7]
Specificity	Formulation buffer shows no dose-response; RS vector shows clear dose-dependence	Criteria met [7]
Dilutional Linearity	R² ≥ 0.85; Slope 0.797-1.049	R² ≥ 0.85 achieved [7]
Intermediate Precision	%GCV <30% for each concentration level	Pooled %GCV: 8.2% [7]
Relative Accuracy	Relative bias within ±15% at all tested levels	All levels within range [7]
Range	50% to 150% of nominal vector concentrations	Demonstrated precision, accuracy, linearity [7]
Robustness	Relative potency 94.3%-112% under varied conditions	RSD for relative potency ≤6.89% [7]

Protocol: Functional T-cell Potency Assay Matrix

For cell therapies with complex MoAs, a single assay is insufficient. The following matrix approach provides comprehensive assessment:

• Cytotoxicity Assay:

  • Co-culture CAR-T cells with target cells expressing the relevant antigen.
  • Measure specific killing using real-time cell impedance (xCELLigence) or flow cytometry-based methods.
  • Quantify over 4-24 hours with multiple effector-to-target ratios.

• Cytokine Release Profiling:

  • Stimulate CAR-T cells with antigen-positive cells or anti-idiotype antibodies.
  • Measure multiple cytokines (IFN-γ, TNF-α, IL-2, IL-6) via multiplex ELISA or Luminex.
  • Profile kinetics over 24-48 hours.

• Phenotypic Characterization:

  • Analyze memory (CCR7, CD45RO), activation (CD25, CD69), and exhaustion (PD-1, LAG-3, TIM-3) markers by flow cytometry.
  • Correlate subsets with functional outcomes.

• Expansion Capacity:

  • Monitor cell proliferation over 7-14 days using dye dilution or cell counting methods.
  • Assess response to repeated antigen stimulation.

Essential Research Reagent Solutions

Implementing robust potency assays requires specialized reagents and tools. The following table details critical materials for cell therapy potency assessment:

Table 4: Essential Research Reagents for Cell Therapy Potency Assays

Reagent Category	Specific Examples	Research Application
Cell-Based Assay Systems	HEK293-LRAT cells [7]	Mechanism-based potency for gene therapies
Cytokine Detection	Multiplex ELISA, Luminex panels [4] [50]	Functional profiling of immune cell products
Flow Cytometry Reagents	CAR detection antibodies, memory/exhaustion marker panels [4] [8]	Phenotypic characterization and identity testing
Molecular Analysis Tools	ddPCR for VCN, NGS for integration sites [4]	Genomic safety and modification assessment
Reference Standards	Well-characterized cell banks, vector preparations [50] [7]	Assay normalization and quality control
Metabolic Assay Kits	Mitochondrial function, glycolytic rate tests [4]	Metabolic fitness assessment

Strategic Framework for Potency Success

The following decision framework illustrates the critical pathway from mechanism of action to successful regulatory submission:

Diagram 2: Strategic Pathway for Potency Assay Development. This framework emphasizes early planning, continuous refinement, and clinical correlation throughout the product development lifecycle [50] [19] [49].

Historical regulatory delays due to inadequate potency assays provide critical learning opportunities for cell therapy developers. The case studies of lifileucel and other products demonstrate that success requires:

• Early Integration of potency strategy beginning in preclinical development [20]
• Matrix Approach employing multiple orthogonal methods to capture complex MoAs [50] [8]
• Clinical Correlation establishing links between assay results and patient outcomes [19]
• Proactive Regulatory Engagement to align on expectations before submission [20] [50]

The evolution from single-parameter tests to comprehensive, mechanism-based potency matrices represents the field's maturation. By learning from past setbacks and implementing robust, predictive potency assays, developers can accelerate the delivery of transformative therapies to patients while meeting rigorous regulatory standards.

In the rapidly advancing field of cell and gene therapy, robust potency assays are indispensable for ensuring product quality, safety, and efficacy. These assays serve as critical quality attributes that measure the biological activity of therapeutic products based on their mechanism of action (MoA) [4]. As therapeutic modalities grow increasingly complex, the development of potency assays that are both quality control (QC)-friendly and scalable has become a significant challenge for researchers and manufacturers. The potency of FDA-approved CAR T-cell products, for instance, is primarily assessed by measuring IFN-γ release in response to target cells, along with other factors such as cell viability and product-specific attributes like CAR expression [4]. However, traditional approaches often lack the comprehensiveness needed to fully characterize modern cell therapy products, driving the need for innovative strategies that balance analytical depth with practical implementability across development and commercial stages.

Comparative Analysis of Current Potency Assay Platforms

Traditional vs. Emerging Potency Assay Approaches

A comparative evaluation of current potency assay platforms reveals distinct advantages and limitations across methodological approaches. Table 1 summarizes the key characteristics of major assay categories used in cell therapy development.

Table 1: Comparison of Potency Assay Platforms for Cell Therapy Products

Assay Category	Key Measured Parameters	Scalability	QC-Friendliness	Key Limitations
Cytokine Release	IFN-γ, TNF-α, IL-2 secretion	High	High	Lacks direct functional information on target cell status; cannot predict long-term persistence [16]
Functional/Cytotoxicity	Tumor killing capacity, serial killing activity	Medium	Medium	More complex to standardize; may require specialized equipment [16]
Genomic	Vector copy number (VCN), vector integration sites, TCR repertoire	Medium	Medium-High	Requires sophisticated instrumentation; data interpretation complexity [4]
Phenotypic	T-cell differentiation states, memory markers	Medium	Medium	Correlation with clinical outcomes may require validation [4]
Multi-omics	Transcriptomic, epigenomic, proteomic profiles	Low	Low	Complex data analysis; currently more suitable for characterization than lot-release [4]

Experimental Evidence: Performance Comparison Data

Recent studies have generated comparative data evaluating the performance of different assay methodologies. Table 2 presents quantitative data from studies directly comparing assay approaches, highlighting the evolving understanding of their predictive value for product performance.

Table 2: Experimental Performance Data for Potency Assay Methods

Assay Method	Detection Limit	Precision (%CV)	Correlation with Clinical Response	Key Supporting Findings
AVATAR Functional Assay	Not specified	Not specified	Predicts long-term serial killing activity	Manufacturing CAR T-cells under TME-mimetic conditions increased stem-like phenotype and enhanced tumor killing capacity [16]
DNA Methylation Profiling	Not specified	Not specified	18 distinct epigenetic loci associated with CR, EFS, and OS	Analysis of 114 CD19 CAR T-cell products identified epigenetic signatures predictive of clinical outcomes [4]
qPCR for Malaria Detection	0.002-30 parasites/μL (varies by assay)	Variable between assays	Not applicable	Assays with high PCR efficiencies outperformed those with low efficiencies in sensitivity, precision, and consistency [51]
ddPCR for VCN	High sensitivity for absolute quantification	<10% (typical for validated methods)	Essential safety parameter	Widely used in quality control laboratories; mandatory for FDA-approved CAR T-cell products [4] [18]

Advanced Methodologies for Enhanced Assay Robustness

Experimental Protocols for Next-Generation Potency Assessment

Protocol 1: AVATAR Platform Functional Potency Assay

The AVATAR platform addresses limitations of conventional cytokine secretion assays by incorporating tumor microenvironment (TME)-mimetic conditions during CAR T-cell manufacturing and testing [16]. The detailed methodology includes:

• CAR T-cell Manufacturing under TME Conditions:

  • Culture primary T-cells under hypoxic (e.g., 1-5% Oâ‚‚) and hyperbaric conditions
  • Transduce with CD19-specific CAR lentiviral vector at optimized MOI
  • Expand cells for 10-14 days with appropriate cytokine support

• Functional Potency Assessment:

  • Co-culture CAR T-cells with target tumor cells at multiple effector-to-target ratios (e.g., 1:1 to 20:1)
  • Measure tumor killing capacity via real-time cell imaging or flow cytometry
  • Assess serial killing activity through repeated challenge experiments
  • Quantify long-term persistence markers through extended culture (14-21 days)

• Phenotypic Characterization:

  • Analyze stem-like memory markers (CD62L, CCR7, TCF1) via flow cytometry
  • Evaluate exhaustion markers (PD-1, TIM-3, LAG-3) pre- and post-activation
  • Measure metabolic fitness through mitochondrial membrane potential and glycolytic capacity assays

This protocol enables accurate prediction of both immediate potency and long-term persistence, addressing critical gaps in conventional potency assays [16].

Protocol 2: Multi-Omics Product Profiling

Comprehensive CAR T-cell product characterization employs a multi-omics approach to identify critical quality attributes beyond conventional parameters [4]:

• Genomic Profiling:

  • Vector copy number (VCN) quantification via droplet digital PCR (ddPCR)
  • Vector integration site analysis using INSPIIRED or EpiVIA pipelines
  • TCR repertoire diversity assessment through bulk and single-cell TCR sequencing

• Epigenomic Analysis:

  • DNA methylation profiling using bisulfite sequencing
  • Chromatin accessibility assessment via ATAC-seq
  • Identification of epigenetic loci associated with clinical outcomes

• Transcriptomic and Proteomic Characterization:

  • Single-cell RNA sequencing for identification of transcriptional subtypes
  • Surface marker profiling to determine differentiation states
  • Cytokine production capacity at single-cell resolution

This multi-parametric approach enables development of tailored potency assays that better reflect product complexity and clinical performance [4].

Quality Control Frameworks for Assay Standardization

Implementing robust QC frameworks is essential for maintaining assay reliability across development stages and manufacturing sites. The navify Quality Performance platform exemplifies this approach through [52]:

• Peer Group Comparison:

  • Statistical analysis of daily QC data against network, country, regional, and global peer groups
  • Real-time performance monitoring with critical test highlighting
  • Six Sigma calculations for determining assay robustness

• Measurement Uncertainty Quantification:

  • Automated calculation of measurement uncertainty as required by ISO 15189
  • Support for clause 7.3.4 (Evaluation of measurement of uncertainty)
  • Compliance with clause 7.3.7.2 (Internal Quality Control) and 3.12 (Interlaboratory Comparison)

• Cross-Platform Harmonization:

  • Comparison of assays across different instruments
  • Ensuring consistency of patient results regardless of analyzer used
  • Support for both Roche and non-Roche controls and instruments in the Plus version

This framework facilitates compliance with regulatory requirements while providing data-driven insights for continuous assay improvement [52].

Visualization of Assay Development Strategies

Workflow for Comprehensive Potency Assessment

The following diagram illustrates an integrated workflow for developing robust, QC-friendly potency assays that capture critical product characteristics:

Multi-Omics Characterization Approach

This diagram outlines the multi-omics characterization strategy essential for comprehensive product understanding:

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful implementation of robust potency assays requires carefully selected reagents and platforms. Table 3 catalogues essential research tools and their applications in potency assay development.

Table 3: Essential Research Reagent Solutions for Potency Assay Development

Reagent/Platform	Primary Function	Key Applications	Considerations for Implementation
Digital Droplet PCR (ddPCR)	Absolute quantification of nucleic acids	Vector copy number determination, viral titer quantification [4] [18]	Provides unparalleled sensitivity and accuracy; essential for quality control
Flow Cytometry Panels	Multi-parameter cell surface and intracellular staining	Immunophenotyping, transduction efficiency, activation markers [4] [18]	Enables comprehensive product characterization; requires careful panel design
Single-Cell RNA Sequencing	High-resolution transcriptomic profiling	Identification of cellular subsets, exhaustion signatures, differentiation states [4]	Reveals product heterogeneity; computationally intensive
Cytokine Detection Assays	Quantification of soluble mediators	IFN-γ, TNF-α, IL-2 measurement as potency indicators [4] [16]	Standardized platforms available; may require multiplexing for comprehensive assessment
AVATAR Platform	Functional assessment under TME-mimetic conditions	Evaluation of tumor killing capacity and persistence [16]	Enhances predictive value for in vivo performance
Metabolic Assay Kits	Measurement of metabolic pathway activity	Evaluation of mitochondrial function, glycolytic capacity [4]	Correlates with T-cell fitness and persistence
Automated Nucleic Acid Extraction	Standardized sample preparation	Consistent DNA/RNA isolation for molecular analyses [51]	Reduces variability and improves reproducibility

The development of QC-friendly and scalable potency assays requires a strategic approach that balances analytical comprehensiveness with practical implementability. As demonstrated through comparative data, traditional methods like cytokine release assays provide scalability but lack predictive value for long-term persistence, while emerging functional and multi-omics approaches offer enhanced insights but present implementation challenges. The optimal strategy involves implementing a matrix of orthogonal assays that collectively reflect the product's mechanism of action, with careful consideration of which parameters are essential for lot-release versus those used for extended characterization. Furthermore, incorporating advanced QC frameworks that enable peer comparison and statistical performance monitoring ensures ongoing assay robustness. As cell therapies continue to evolve, potency assay strategies must similarly advance through integration of technological innovations while maintaining the rigor required for regulatory compliance and patient safety.

Ensuring Regulatory Success: A Roadmap for Potency Assay Validation and Comparability

For cell therapy products (CTPs), demonstrating potency—the quantitative measure of biological activity—is a fundamental regulatory requirement for product release and stability testing [9] [8]. The potency assay stands as the sole quality control test that measures the function of the product, making it a cornerstone of Chemistry, Manufacturing, and Control (CMC) activities. The ICH Q2(R2) guideline, "Validation of Analytical Procedures," provides the foundational framework for establishing the performance characteristics of these critical assays, ensuring they are suitable for their intended purpose throughout the product lifecycle [53]. For developers of CTPs, navigating the intersection of ICH Q2(R2)'s validation principles with the complex biological nature of living medicines presents a unique set of challenges and requires a science-driven, rational approach.

ICH Q2(R2) Validation Framework for Analytical Procedures

The objective of analytical procedure validation, as defined by ICH Q2(R2), is to demonstrate that the procedure is suitable for its intended purpose. For a potency assay, this translates to providing a reliable measure of the biological activity linked to the product's clinical efficacy.

Key Performance Characteristics and Their Definitions

The table below summarizes the key performance characteristics required for a validated potency assay as outlined in ICH Q2(R2) [53].

Table 1: Key Performance Characteristics for Potency Assay Validation per ICH Q2(R2)

Performance Characteristic	Description
Accuracy	The closeness of agreement between the measured value and an accepted reference value.
Precision	The degree of agreement among individual test results under prescribed conditions. This includes repeatability (intra-assay precision) and intermediate precision (variation between days, analysts, or equipment).
Specificity	The ability to assess the analyte unequivocally in the presence of other components, including impurities or matrix elements.
Working Range	The interval between the upper and lower levels of analyte that have been demonstrated to be determined with suitable levels of precision, accuracy, and linearity.
Linearity	The ability of the assay to produce results that are directly proportional to the concentration of the analyte.

Application of ICH Q2(R2) to Cell Therapy Potency Assays

Applying this framework to CTPs requires careful consideration of the product's complexity and mechanism of action (MOA). The validation strategy must be tailored to the specific technology platform, such as flow cytometry, bioassays, or molecular methods. The overarching goal is to ensure the assay can detect changes in the product's biological function, thereby serving as a stability-indicating assay as required by ICH Q5C [53].

Current Landscape of Potency Assays for Approved Cell Therapies

An analysis of the 31 US FDA-approved CTPs from 2010 through 2024 provides a concrete view of real-world potency assay strategies. A total of 104 potency tests are reported for these products, with an average of 3.4 tests per CTP [9] [8].

Categorization of Potency Assay Measurements

The 71 non-redacted potency tests can be categorized into five main types of measurements, as shown in the table below. Many products employ a combination of tests to fully characterize potency [8].

Table 2: Categories of Potency Measurements for US FDA-Approved Cell Therapy Products

Assay Category	Number of Tests	Percentage	Example Methods
Viability and Count	37	52%	Total nucleated cells, viable CD34+ cell count, cell viability [9]
Expression	19	27%	CAR expression by flow cytometry, anti-CD19 CAR expression [9]
Bioassays	7	7%	Interferon-γ production upon antigen stimulation, cytotoxic activity [9]
Genetic Modification	6	9%	Vector copy number (qPCR), percent LVV+ cells [9]
Histology	2	3%	Tissue organization, viability & retention of important cell types [9]

This data shows a strong reliance on physicochemical tests like "Viability and Count" and "Expression." However, it is notable that 32% of potency tests across these products are redacted in regulatory documents, suggesting that proprietary, and potentially more complex, bioassays are also widely used [9] [8].

Comparative Analysis of Potency Assay Platforms

Selecting the appropriate assay platform is a critical decision in CTP development. The choice often involves a balance between technological simplicity and biological relevance.

Comparison of Key Assay Formats

The table below provides a comparative analysis of common potency assay platforms used in the development of CTPs.

Table 3: Comparative Analysis of Cell Therapy Potency Assay Platforms

Assay Platform	Description	Proximity to MOA	Throughput	Regulatory Fit	Example Use Case
Surrogate Assay (e.g., ELISA)	Measures a single protein interaction or concentration.	Low	High	Accepted in early phases with scientific rationale [53]	Quantifying CAR expression level [9]
Cell-Based Bioassay (2D)	Measures a functional response in a planar cell culture.	Medium	Medium	Common; may lack clinical predictive power [54]	IFN-γ release in response to target cells [9]
Advanced Bioassay (3D)	Measures a functional response in a 3D microphysiological system.	High	Low	Emerging; demonstrates improved clinical prediction [54]	Predicting patient pain relief in osteoarthritis trial [54]
Flow Cytometry	Measures cell surface or intracellular protein expression.	Low-Medium	High	Well-established for identity/phenotype [9]	Viable CD34+ cell count, CAR+ percentage [9]

A key development in the field is the emergence of advanced 3D potency assays. For example, a microfluidic on-chip 3D system used to evaluate bone marrow aspirate concentrate (BMAC) for osteoarthritis demonstrated elevated levels of immunomodulatory and trophic proteins compared to 2D culture. Most importantly, this 3D assay showed a higher correlative power with patient pain scores in a phase 3 clinical trial, establishing a potency assay with improved predictive power for clinical outcomes [54].

Experimental Protocol for a Predictive 3D Potency Assay

The following detailed methodology is adapted from a study establishing an on-chip 3D potency assay for a BMAC cell therapy [54].

Protocol: On-Chip 3D Potency Assay for Bone Marrow Aspirate Concentrate (BMAC)

Objective: To assess the secretory potency of BMAC cells in a 3D microphysiological system and correlate the secreted analyte profile with clinical outcomes.

Materials and Reagents:

• Poly(dimethylsiloxane) (PDMS) Microfluidic Device: Provides the structure for the 3D culture and media perfusion.
• PEG-4MAL Hydrogel: A 4-arm maleimide-functionalized poly(ethylene-glycol) synthetic hydrogel for 3D cell encapsulation.
• Cell-Adhesive RGD Peptide: Incorporated into the hydrogel to support cell adhesion.
• Protease-Degradable Peptide: A cross-linker for the hydrogel, allowing cell-mediated remodeling.
• OA Simulated Syncytial Fluid (simSF): A culture media supplement mimicking the protein composition and viscosity of osteoarthritic synovial fluid.
• Multiplexed Cytokine/Chemokine Assay (e.g., Luminex): For quantifying secreted analytes.

Methodology:

• Cell Encapsulation: Thaw cryopreserved BMAC cells and encapsulate them in the PEG-4MAL hydrogel pre-mixed with RGD peptide and cross-linkers. The hydrogel formulation supports high viability and secretory activity of key cell populations like mesenchymal stromal cells.
• Device Perfusion: Incorporate the cell-laden hydrogel into the microfluidic device and perfuse with media at a rate of 1.0 µL/min for 24 hours. This perfusion rate is within the physiological range of interstitial fluid velocity.
• Condition Testing: Culture cells in two conditions: (i) basal control media (ctrl) and (ii) media supplemented with 10% simSF (simSF). The simSF condition provides a more disease-relevant microenvironment.
• Secreted Analyte Analysis: After 24 hours, collect the perfused media and analyze it using a multiplexed assay to quantify the levels of 24 pre-selected immunomodulatory and trophic proteins (e.g., cytokines, chemokines, MMPs).
• Data Modeling and Correlation: Use the secreted analyte levels in a data-driven linear regression prediction model. The model is cross-validated using patient-matched clinical outcome data (e.g., pain scores) to establish a correlation between the in vitro potency assay and clinical efficacy.

The Scientist's Toolkit: Essential Reagents for Potency Analysis

The table below details key research reagent solutions essential for developing and executing robust potency assays for cell therapies.

Table 4: Essential Reagent Solutions for Cell Therapy Potency Assays

Reagent / Material	Function in Potency Assay
Defined Hydrogel (e.g., PEG-4MAL)	Provides a synthetic, reproducible 3D extracellular matrix for cell encapsulation, supporting more physiologically relevant cell function than 2D plastic [54].
Engineered Cell Lines (Target/Reporter)	Used as biological substrates in bioassays to measure a specific functional response (e.g., cell killing, cytokine release) upon interaction with the CTP [53].
Cytokine/Chemokine Multiplex Panels	Enable simultaneous quantification of dozens of secreted analytes from a small sample volume, providing a rich secretory profile for potency assessment [54].
Validated Critical Reagents (e.g., Antibodies)	High-quality, well-characterized antibodies for flow cytometry or ELISA are essential for obtaining precise, accurate, and reproducible measurements of cell surface markers [9] [53].
Mechanistically Relevant Stimuli (e.g., simSF)	Disease-relevant culture media supplements that challenge the CTP to elicit a functional response that correlates with its in vivo mechanism of action [54].

Logical Workflow for Potency Assay Development and Validation

The following diagram illustrates the critical stages of developing and validating a potency assay, from initial concept to regulatory filing, in alignment with ICH Q2(R2) and product lifecycle management.

Diagram 1: Potency assay development workflow.

The successful development and validation of a potency assay for a cell therapy product is a multi-stage process that demands a deep understanding of the product's MOA, a strategic application of the ICH Q2(R2) guidelines, and careful selection of appropriate assay technologies. While current approved products often leverage a matrix of tests heavily weighted toward viability, count, and expression assays, the future lies in developing more predictive, functionally relevant bioassays. The emergence of advanced 3D microphysiological systems as clinically predictive tools marks a significant step forward. By building a scientifically rigorous bridge between a product's biological function and its clinical performance, developers can create robust potency assays that not only meet regulatory requirements but also ensure that every batch of a cell therapy is safe, effective, and consistently potent for patients.

In the development and quality control of cell therapies, potency assays are a legal requirement for lot release testing, serving as a vital measure of the product's biological activity and therapeutic potential [3] [8]. These assays provide a quantitative link between the product's critical quality attributes (CQAs) and its intended mechanism of action (MoA) [55]. Given the complex and living nature of cell-based advanced therapy medicinal products (ATMPs), ensuring their consistency, safety, and efficacy presents unique challenges [5]. The reliability of any potency assay hinges on the rigorous validation of its core performance parameters. This guide objectively compares the established benchmarks for Specificity, Linearity, Precision, and Accuracy, based on regulatory guidelines and their practical application in the field of cell therapy.

Defining the Core Parameters

Specificity

Specificity is the ability of an analytical procedure to assess the analyte unequivocally in the presence of other components that may be expected to be present, such as impurities, degradants, or matrix components [56]. In the context of cell therapies, this confirms that the measured signal is solely due to the intended biological activity.

Linearity and Range

Linearity defines the ability of an assay to obtain test results that are directly proportional to the concentration (or amount) of the analyte in the sample within a given Range [57] [56]. The range is the interval between the upper and lower concentration levels of the analyte for which suitable levels of precision, accuracy, and linearity have been demonstrated [56].

Accuracy

Accuracy expresses the closeness of agreement between the test result obtained by the method and the true value (or an accepted reference value) [57]. It is a measure of systematic error, often reported as percent recovery of a known, spiked amount of analyte [56].

Precision

Precision describes the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions [57]. It is a measure of random error and is typically considered at three levels:

• Repeatability: Precision under the same operating conditions over a short time interval [57] [56].
• Intermediate Precision: Variation within a laboratory due to random events like different days, analysts, or equipment [57] [56].
• Reproducibility: Precision between different laboratories, often assessed in collaborative studies [57] [56].

Table 1: Summary of Core Performance Parameters and Their Definitions

Parameter	Core Definition	Key Aspect Measured
Specificity	Ability to measure analyte unequivocally in the presence of potential interferents [56].	Selectivity of the assay.
Linearity	Ability to produce results directly proportional to analyte concentration [57] [56].	Proportionality of response.
Range	The interval between upper and lower analyte concentrations for which linearity, accuracy, and precision are demonstrated [56].	Validated working interval.
Accuracy	Closeness of agreement between the measured value and the true value [57].	Systematic error (bias).
Precision	Closeness of agreement between a series of measurements from multiple samplings [57].	Random error (variance).

Performance Benchmarks and Comparison

The acceptance criteria for these parameters vary depending on the type of assay (e.g., biochemical, cell-based) and its specific application. The following section compares typical benchmarks derived from regulatory guidelines and real-world case studies in cell therapy.

Quantitative Comparison of Validation Criteria

Table 2: Comparative Acceptance Criteria for Performance Parameters

Parameter	Typical Acceptance Criteria	Common Experimental Approach (Drug Substance/Product)	Case Study Example (AAV2-hRPE65v2 Potency Assay) [7]
Specificity	No dose-response from blank/inactive components; target must show clear, distinguishable response [56] [7].	Compare analyte response in presence and absence of excipients/impurities [56].	Formulation buffer showed no dose-response; reference standard and test article demonstrated overlapping, parallel dose-response curves.
Linearity	R² > 0.95 (for linear regression); residual sum of squares should be low [57] [56].	Minimum of 5 concentration points across the specified range [57] [56].	Demonstrated for square root-transformed data; R² ≥ 0.85 for dilutional linearity of relative potency.
Range	Assay: 80-120% of test concentration; Content Uniformity: 70-130% [57] [56].	Confirm linearity, accuracy, and precision at the extremes and within the range [56].	Range established from 50% to 150% of nominal vector concentration.
Accuracy	Reported as % recovery; bias should be within ±15% for bioassays [57] [7].	Minimum of 9 determinations over 3 concentration levels (3 replicates each) [57] [56].	Relative bias within ±15% at all tested levels (e.g., +5.9% at 50%, -0.1% at 100% potency).
Precision	Repeatability: ≤ 6 determinations at 100% concentration [56].Intermediate Precision: %GCV < 30% for cell-based assays [57] [7].	Repeatability: 9 determinations over specified range or 6 at 100% [56]. Intermediate Precision: Vary days, analysts, equipment [57].	Intermediate Precision: Pooled %GCV of 8.2%; individual levels ranged from 4.5% to 9.7%.

Analysis of Parameter Interdependence

These core parameters are not independent. The validation of Range is derived from demonstrations of Linearity, Accuracy, and Precision [56]. Furthermore, according to ICH guidelines, Accuracy may be inferred once Precision, Linearity, and Specificity have been established [57]. This interconnectedness underscores the need for a holistic validation strategy rather than treating each parameter in isolation.

Experimental Protocols for Determination

Protocol for Specificity and Selectivity

• Sample Preparation: For a drug product, prepare a synthetic mixture of the drug product components to which known quantities of the drug substance have been added. Alternatively, spike known quantities of the analyte into the drug product [57].
• Interference Testing: Analyze samples containing potential interferents (e.g., formulation buffer, inactive product components) using the proposed analytical procedure. The response from these should be negligible [7].
• Comparison: The results from the spiked sample should be compared to those from an unspiked sample or a second, well-characterized orthogonal procedure [57] [56].

Protocol for Linearity and Range

• Solution Preparation: Prepare a minimum of 5 concentrations of the analyte, appropriately distributed across the intended range (e.g., 50%, 75%, 100%, 125%, 150%) [57] [7]. This can be done by diluting a standard stock solution or by separate weighings of synthetic mixtures [56].
• Analysis: Analyze each concentration level according to the analytical procedure.
• Data Analysis: Plot the measured signal (response) against the analyte concentration. Evaluate the linear relationship by visual inspection and by statistical analysis, calculating a regression line (e.g., via least squares). Report the correlation coefficient (R²), y-intercept, slope, and residual sum of squares [57] [56].

Protocol for Accuracy

• Spiking Design: Conduct a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g., 3 concentrations with 3 replicates each) [57] [56].
• For Drug Product: Add known quantities of the analyte to a synthetic matrix (or the drug product itself if matrix recreation is difficult) [57].
• Calculation: Accuracy is reported as percent recovery by the assay of the known added amount of analyte, or as the difference between the mean and the accepted true value along with confidence intervals [57] [56].

Protocol for Precision (Repeatability & Intermediate Precision)

• Repeatability: Under the same operating conditions, analyze either: a) a minimum of 9 determinations covering the specified range (e.g., 3 concentrations/3 replicates each), or b) a minimum of 6 determinations at 100% of the test concentration [57] [56].
• Intermediate Precision: Incorporate variations into the study design, such as different days, different analysts, and different equipment. The use of an experimental design (matrix) is encouraged [57] [56].
• Data Reporting: For each type of precision, report the standard deviation, relative standard deviation (coefficient of variation), and confidence interval [57].

Visualizing the Validation Workflow and Relationships

The following diagram illustrates the logical workflow for establishing and interconnecting these core performance parameters, from initial definition to the final control strategy.

Figure 1. Logical workflow for assay validation, showing interdependence of core parameters. Specificity is foundational, while Linearity, Accuracy, and Precision are jointly used to define the validated Range.

The Scientist's Toolkit: Essential Research Reagent Solutions

The successful execution of potency assays, especially in a GMP environment, relies on a suite of critical reagents and tools.

Table 3: Key Research Reagent Solutions for Potency Assay Development

Tool / Reagent	Function in Potency Assay	Application Example
Reference Standard (RS)	A well-characterized drug lot of known potency; serves as the benchmark for calculating Relative Potency (%RP) of test samples, controlling inter-assay variability [3] [7].	Used in parallel-line analysis for AAV vector potency [7].
Cell-Based Assay Systems	Model the biological mechanism of action (MoA) of the therapy; used in functional potency assays (e.g., cytotoxicity, signaling activation) [5] [58].	HEK293-LRAT cells used to measure enzymatic activity of AAV2-hRPE65v2 [7].
Cytokine Detection Assays	Measure surrogate markers of cell activity (e.g., IFN-γ, TNF-α) as an indicator of potency for T-cell therapies [4] [5].	IFN-γ release measured in response to target cells for CAR T-cell products [4].
Flow Cytometry Reagents	Identify and quantify cell surface and intracellular markers (e.g., CAR expression, differentiation markers) for identity and potency testing [4] [8].	"CAR expression from viable T cells" is a common potency test for FDA-approved CAR-T products [8].
Real-Time Cell Analysis (RTCA)	Label-free, impedance-based technology to monitor cell health and cytolytic activity in real-time, providing kinetic potency data (e.g., KT50) [58].	Used to monitor NK92 cell-mediated killing of cancer cell lines [58].
LC-MS/MS Systems	Provide highly specific and quantitative readouts for analytes, especially in assays measuring specific enzymatic products or metabolites [7].	Quantification of 11-cis-retinol in the potency assay for AAV2-hRPE65v2 [7].

The rigorous validation of specificity, linearity, precision, and accuracy forms the cornerstone of reliable and meaningful potency assays for cell therapies. As shown by the data from approved products, a combination of simple, robust tests (like viability and expression) and complex, MoA-reflective bioassays is often employed to ensure product quality [8]. The benchmarks and protocols outlined in this guide provide a framework for developers. Adherence to these core performance parameters, guided by regulatory standards and a quality-by-design approach, is essential for demonstrating that a potency assay is fit-for-purpose, ensuring the consistent delivery of safe and efficacious cell therapies to patients.

Demonstrating Comparability for Manufacturing Process Changes

In the rapidly advancing field of cell therapy, manufacturing process changes are often necessary to improve scalability, cost-effectiveness, and product quality. However, these modifications require rigorous comparability assessment to ensure they do not adversely impact the critical quality attributes (CQAs) of the final therapeutic product. For cell-based therapies, demonstrating comparability following manufacturing changes presents unique challenges due to the complex, living nature of these products and the limited historical data available, particularly during accelerated development pathways [59]. This guide examines the experimental frameworks and analytical methodologies essential for objectively demonstrating comparability while maintaining product safety, potency, and efficacy.

Critical Quality Attributes in Cell Therapy Comparability Assessment

Key Product Characteristics for Evaluation

Table 1: Critical Quality Attributes for Cell Therapy Comparability Studies

Attribute Category	Specific Parameters	Analytical Methods	Acceptance Criteria
Potency	Cytokine secretion (IFN-γ, TNF-α, IL-2), cytotoxic activity, tumor killing capacity	ELISA, multiplex immunoassays, flow cytometry, cytotoxicity assays	No significant reduction in potency; maintained dose-response relationship
Phenotype	T-cell subpopulations (naïve, stem-cell memory, central memory, effector memory), exhaustion markers (PD-1, TIM-3), activation markers	Multicolor flow cytometry (14+ colors), immunophenotyping	Consistent distribution of critical subpopulations; no emergence of aberrant phenotypes
Genetic Stability	Vector copy number, integration sites, TCR repertoire diversity	ddPCR, next-generation sequencing (INSPIIRED, EpiVIA pipelines)	Maintained within established safety limits; consistent integration profile
Functional Persistence	Long-term serial killing, expansion capacity, resistance to exhaustion	AVATAR platform, repeated stimulation assays, in vivo models	Sustained functional activity through multiple challenges
Viability & Expansion	Post-thaw viability, expansion kinetics, doubling time	Trypan blue exclusion, automated cell counting, metabolic assays	Meets established manufacturing specifications

Experimental Design Strategies for Comparability Assessment

Statistical Approaches for Comparability Studies

Design of Experiments (DOE) provides a structured framework for comparability assessment by systematically evaluating multiple factors and their interactions simultaneously rather than through traditional one-factor-at-a-time approaches [60] [61]. This methodology is particularly valuable for complex cell therapy processes where multiple parameters may interact to affect product CQAs.

The DOE process typically follows six key steps: (1) establishing clear objectives for the comparability study; (2) selecting critical factors, levels, and response variables; (3) designing the experiment with appropriate controls; (4) conducting randomized experimental runs; (5) analyzing data using statistical methods; and (6) implementing findings based on evidence-based conclusions [61]. For cell therapy applications, fractional factorial designs are often employed due to the large number of factors involved, allowing efficient investigation of the most significant parameter interactions without requiring exhaustive experimentation [61].

Industry Standards for Comparability Protocol Development

According to industry best practices, manufacturers should evaluate over 140 commonly encountered process, material, and analytical changes derived from FDA guidance on Chemistry, Manufacturing, and Control Changes to an Approved Application [59]. The comparability assessment strategy should be risk-based, with the depth of analysis commensurate with the stage of clinical development (pre-pivotal vs. pivotal) and the potential impact of the change on product quality and safety.

Advanced Analytical Methods for Comparability Assessment

Multi-Omics Profiling Technologies

Table 2: Advanced Analytical Methods for Comprehensive Product Characterization

Technology Platform	Applications in Comparability	Key Readouts	Regulatory Considerations
Single-cell RNA-seq	Transcriptomic profiling, identification of novel subsets, exhaustion signatures	Differential gene expression, unique molecular identifiers, pathway analysis	Standardization of protocols, validation of reproducibility
Epigenetic profiling	DNA methylation analysis, chromatin accessibility (ATAC-seq), differentiation state	Methylation patterns at critical loci (18 sites associated with clinical response)	Correlation with persistence and potency
TCR repertoire sequencing	Clonal diversity assessment, tracking of persistent clones, γδ T-cell detection	Clonality metrics, highly expanded T-cell clones, VDJ usage	Monitoring for oligoclonality and restricted diversity
Proteomic analysis	Surface marker expression, intracellular signaling proteins, cytokine production	Protein abundance, post-translational modifications, activation states	Linkage to functional potency outcomes
Metabolic profiling	Glycolytic activity, mitochondrial fitness, oxidative stress response	Metabolic flux, ATP production, nutrient utilization	Relationship to differentiation and persistence

Functional Potency Assays

Beyond characterization of cellular attributes, functional assays that measure biological activity are fundamental to comparability assessment. Conventional cytokine secretion assays (measuring IFN-γ, TNF-α, and IL-2) provide limited information about long-term functionality and persistence [16]. Next-generation functional assays incorporate models of the tumor microenvironment (TME), including hypoxic and hyperbaric conditions, to better predict in vivo performance [16]. These advanced platforms can identify enhanced stem-like phenotypes and improved serial killing capacity in modified manufacturing processes, providing critical data for comparability determination.

For engineered T-cell therapies like CAR-T products, potency assessments should evaluate immediate effector functions (cytotoxicity, cytokine release, degranulation), viability and expansion potential, and persistence characteristics [4]. A comprehensive matrix of assays is recommended to fully profile these key activities, as no single assay can adequately capture the complex mechanism of action of living cell products.

Case Study: Manufacturing Process Modification for NY-ESO-1 Targeted T-Cell Therapy

Experimental Protocol for Process Comparability

A published case study on affinity-enhanced TCR therapy for NY-ESO-1 and LAGE-1 antigens provides a practical example of comparability assessment following manufacturing changes [62]. The modified process involved changes to: (A) manipulation of incoming apheresis product, (B) initial T-cell enrichment methods, (C) medium components, and (D) final formulation.

Experimental Methodology:

• Parallel Manufacturing: Both pre-change and post-change processes were run concurrently using donor-matched starting material to minimize biological variability.
• Comprehensive Phenotyping: 14-color flow cytometry panels were employed to analyze T-cell subpopulations, differentiation states, and activation/exhaustion markers.
• Functional Assessment: Cytokine expression profiles (IFN-γ, IL-2, TNF-α) were measured following antigen-specific stimulation.
• Culture Expansion: Products from both processes underwent prolonged in vitro expansion to assess long-term growth characteristics and stability.

Results and Interpretation: The comparability study demonstrated equivalent phenotype distributions across critical T-cell memory subsets, consistent cytokine expression profiles, and comparable expression of exhaustion markers between the pre-change and post-change products. This comprehensive analysis supported the conclusion of comparability while providing detailed characterization data for regulatory submission.

The Scientist's Toolkit: Essential Research Reagents and Platforms

Table 3: Key Research Reagent Solutions for Comparability Assessment

Reagent/Platform	Primary Application	Function in Comparability Studies
AVATAR Cell Culture System (Xcellbio)	Tumor microenvironment modeling	Enables functional assessment under physiologically relevant hypoxic and hyperbaric conditions
Droplet Digital PCR (Bio-Rad)	Vector copy number quantification	Provides absolute quantification of transgene copy number with superior sensitivity and accuracy
INSPIIRED Pipeline	Viral integration site analysis	Detects vector integration events at bulk-cell resolution for safety assessment
EpiVIA Pipeline	Single-cell integration analysis	Enables detection of integration sites at single-cell resolution
14+ Color Flow Cytometry Panels	Immunophenotyping	Simultaneous analysis of multiple T-cell subsets, differentiation states, and exhaustion markers
Single-cell RNA-seq Platforms	Transcriptomic profiling	Identification of novel cellular subsets and molecular signatures associated with product potency
Multiplex Cytokine Assays	Functional potency assessment	Simultaneous measurement of multiple inflammatory mediators and effector molecules
TCR Sequencing Reagents	Clonality assessment	Evaluation of TCR repertoire diversity and tracking of persistent clones

Regulatory Considerations and Industry Perspectives

Demonstrating comparability for cell-based therapies following manufacturing changes requires careful planning and execution of studies designed to detect potential impacts on CQAs. Regulatory agencies expect a science-based, risk-adjusted approach that considers the stage of product development and the potential impact of the change on product safety and efficacy [59]. The comparability exercise should be viewed as an iterative process that may require additional data from clinical studies to fully resolve residual uncertainty, particularly for changes implemented during late-stage development.

Industry perspectives emphasize that successful comparability demonstration requires early engagement with health authorities, statistically sound study designs, and comprehensive product characterization using orthogonal methods [59]. The evolving regulatory landscape for advanced therapies necessitates transparent communication about manufacturing changes and their potential impact on product quality, particularly as the industry moves toward commercial-scale production.

Within the critical field of cell and gene therapy development, potency assays stand as a fundamental component of quality control, required by regulatory authorities to ensure that products possess the intended biological activity for clinical efficacy [63] [64]. For advanced therapies, the development of a robust, quantitative potency assay that reflects the product's mechanism of action (MoA) is one of the most significant analytical challenges [65]. This case study examines the validation of a specific quantitative cell-based relative potency assay for Voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus (AAV) serotype 2 vector. As the first gene therapy approved in the United States for a genetic disease, the Luxturna program established a benchmark for AAV vector potency testing [63] [7]. We will explore the experimental protocols, validation parameters, and quantitative data supporting this assay, and situate its approach within the broader context of potency testing for biological products, including a comparative analysis with other approved cell therapy products (CTPs).

The Luxturna AAV2-hRPE65v2 Vector and Its Mechanism of Action

Luxturna is an AAV2 vector encoding the human retinal pigment epithelium-specific 65 kDa protein (RPE65) cDNA, developed to treat visual impairment from biallelic RPE65 mutations [63]. The biological activity of the vector depends on a multi-step mechanism of action: successful gene transfer to target cells, followed by transcription and translation of the RPE65 transgene, resulting in the production of functional RPE65 protein. This enzyme acts as a retinoid isomerohydrolase, executing a crucial step in the visual cycle by converting all-trans-retinol (at-ROL) to 11-cis-retinol (11-cis-ROL) [63] [7]. The validated potency assay was designed to quantitatively measure the output of this final, functionally critical enzymatic step.

Experimental Protocol for the AAV2-hRPE65v2 Potency Assay

Assay Workflow and Design

The assay was designed as a cell-based relative potency method, aligning with US Pharmacopeia (USP) <1032> guidelines [63] [7]. The following workflow was established and optimized:

• Cell Line and Transduction: HEK293 cells, which are efficiently transduced by AAV2 and lack endogenous RPE65 expression, were genetically modified to stably express lecithin retinol acyltransferase (LRAT). This modification was essential to support the visual cycle pathway. These HEK293-LRAT cells were transduced with the AAV2-hRPE65v2 vector across nine different multiplicities of infection (MOIs), spanning a range from 1×10⁴ to 1.28×10⁶ vector genomes (vg) per cell [63] [7].
• Lysate Preparation and Enzymatic Reaction: After transduction, cell lysates were prepared. The protein concentration of these lysates was quantified to normalize the subsequent enzymatic reaction. The lysates were then incubated with the substrate, all-trans-retinol (at-ROL), and cellular retinaldehyde binding protein (CRALBP) to facilitate the conversion to the product, 11-cis-retinol (11-cis-ROL) [63].
• Quantitative Detection and Analysis: The 11-cis-ROL product was separated from the substrate and other retinoid isomers using liquid chromatography (LC) and accurately quantified using tandem mass spectrometry (LC-MS/MS). The resulting dose-response curve, plotting 11-cis-ROL concentration against the log10(MOI), was fitted using a three-parameter logistic (3PL) model. The relative potency of a test article was determined by comparing its dose-response curve to that of a reference standard using parallel line analysis (PLA) software [63] [7].

The diagram below illustrates this integrated experimental workflow and the biological mechanism it measures.

Key Research Reagent Solutions

The successful execution of this potency assay relied on several critical reagents and cell lines, each serving a specific function to ensure the assay's relevance and reliability.

Table: Essential Research Reagents for the AAV Potency Assay

Reagent / Cell Line	Function in the Assay
HEK293-LRAT Cell Line	Engineered host cells that are efficiently transduced by AAV2 and provide the necessary LRAT enzyme to support the visual cycle, enabling the RPE65 enzymatic reaction.
AAV2-hRPE65v2 Reference Standard (RS)	A well-characterized vector batch with a defined potency of 100%, serving as the benchmark for comparing the relative potency of test articles.
All-trans-Retinol (at-ROL)	The natural, non-radioactive substrate for the RPE65 isomerohydrolase enzyme. Its use avoids the safety and facility requirements associated with radioactive isotopes.
CRALBP (Cellular Retinaldehyde Binding Protein)	A binding protein that facilitates the enzymatic reaction in the assay, supporting the conversion of at-ROL to 11-cis-ROL.
LC-MS/MS System	The analytical platform used for the specific separation, detection, and highly accurate quantification of the 11-cis-ROL product, providing a quantitative readout.

Assay Validation Data and Acceptance Criteria

To meet regulatory requirements for a commercial product, the assay underwent rigorous validation, evaluating seven key performance characteristics as per USP <1033> [7]. The validation was conducted across five relative potency levels (50%, 75%, 100%, 125%, and 150%) over 20 independent assays. The results are summarized in the table below.

Table: Validation Parameters and Results for the AAV2-hRPE65v2 Potency Assay

Validation Parameter	Experimental Design	Acceptance Criteria	Validation Result
System & Sample Suitability	3PL model fit with 9 MOIs; 90% CI for relative potency.	CI within 76%-130%; equivalence tests must pass.	Consistently met across 20 assays.
Specificity	Test formulation buffer (no vector) and RS vector.	Buffer shows no dose-response; RS shows clear dose-response.	No activity from buffer; specific response from RS confirmed.
Dilutional Linearity	Test articles at 50%, 75%, 100%, 125%, 150% potency.	R² ≥ 0.85; slope 0.797–1.049.	R² = 0.99; slope = 0.92; intercept = 0.08 [7].
Intermediate Precision	% Geometric Coefficient of Variation (%GCV) across analysts, days.	<30% for each potency level.	Pooled %GCV = 8.2% (range: 4.5% @50% to 9.7% @150%) [7].
Relative Accuracy	Relative bias of measured vs. expected potency.	Within ±15% at all levels.	All levels within criteria (e.g., 100%: -0.1%, 150%: -7.2%) [7].
Range	MOIs from 1×10⁴ to 1.28×10⁶ vg/cell.	Demonstrate precision, accuracy, linearity within range.	50% to 150% of nominal concentration established as valid range.
Robustness	Deliberate variations in transduction time, sonication pulses, CRALBP concentration.	Assay remains valid; RSD for potency ≤6.89%.	Relative potency 94.3%–112%; RSD ≤6.89% under varied conditions [7].

The following diagram outlines the logical framework of the validation process, linking the tested parameters to the overall goal of ensuring assay quality and reliability.

Comparative Analysis with Other Potency Assay Approaches

Comparison with Another AAV Vector Potency Assay

The approach for Luxturna can be contrasted with a potency assay developed for an AAV8 vector expressing human uridine diphosphoglucuronosyl transferase 1A1 (AAV8-hUGT1A1) to treat Crigler-Najjar syndrome [64]. This assay also utilized a cell-based system but employed human hepatoma (Huh7) cells and two distinct readouts: transduction efficiency, quantified by flow cytometry to detect UGT1A1-positive cells, and transgenic activity, measured by a bilirubin conjugation assay [64]. Both the Luxturna and AAV8-hUGT1A1 assays demonstrated a linear dose-response (R² > 0.98) and adequate intra-assay and inter-day precision (CV% < 23%), confirming that cell-based assays with functional readouts can robustly translate to in vivo efficacy [64].

Potency Assay Landscape for FDA-Approved Cell Therapy Products

An analysis of the 31 FDA-approved Cell Therapy Products (CTPs) through 2024 provides a broader context for potency testing strategies. This analysis found that these products use an average of 3.4 potency tests per CTP, with "Viability and count" (52%) and "Expression" (27%) being the most common test types [8] [9] [66]. For example, all seven approved CAR-T products use flow cytometry to measure CAR expression, and most also employ a bioassay, such as interferon-gamma (IFN-γ) release upon target cell stimulation, to quantify effector function [8] [9]. The Luxturna assay, with its single, highly quantitative LC-MS/MS readout of a specific enzymatic function, represents a different and highly mechanistic approach within this spectrum.

Table: Comparison of Potency Assay Types for Approved Biologics

Product Category	Common Potency Assay Types	Key Readouts	Relation to MoA
AAV Vectors (e.g., Luxturna)	Cell-based biochemical activity.	Quantitative enzymatic product formation (LC-MS/MS).	Directly measures final, critical biochemical output.
CAR T-Cells (e.g., Kymriah, Yescarta)	Cytokine release bioassay, Cell surface expression.	IFN-γ release upon antigen stimulation, CAR+ % by flow cytometry.	Measures proximal T-cell activation and effector function.
Hematopoietic Stem Cells (Cord Blood)	Viability and count, Colony-forming units (CFU).	CD34+ cell count, viable nucleated cells, CFU.	Serves as a surrogate for engraftment and differentiation potential.

The validation of the quantitative cell-based relative potency assay for AAV2-hRPE65v2 represents a paradigm for AAV vector lot release and stability testing. Its success is rooted in the direct linkage to the product's mechanism of action, specifically the critical enzymatic function of the transgene [63]. The adoption of a relative potency methodology, benchmarked against a reference standard, effectively controls for the inherent variability of biological systems, as encouraged by USP <1032> [63] [7].

This case study highlights several best practices for potency assay development: the importance of a quantitative, mechanism-based readout; the necessity of a comprehensive validation plan addressing precision, accuracy, and robustness; and the strategic advantage of implementing a relative potency model. Furthermore, the move from an initial, cumbersome in vivo model in rd12 mice to this streamlined in vitro assay underscores a broader industry trend toward developing robust, reproducible, and efficient quantitative assays that reduce animal use and accelerate testing timelines without compromising quality [63] [64].

In the broader context of cell therapy potency assays research, this case study demonstrates that while the specific assay format must be product-specific, the fundamental principles of linking the readout to the mechanism of action and conducting rigorous validation are universal. As the field advances with more complex products, the lessons from this pioneering AAV potency assay will continue to inform the development of next-generation potency methods for both autologous and allogeneic therapies [4] [12].

Conclusion

The development of robust, mechanism-of-action-relevant potency assays is not merely a regulatory hurdle but a fundamental pillar of successful cell therapy development. As the field advances, the integration of potency testing early in the development process, coupled with a strategic approach to validation and the adoption of novel tools like standardized reference materials and multi-omics profiling, is crucial for de-risking programs and accelerating timelines. The future of potency testing will likely involve increasingly sophisticated assay matrices that collectively capture the complex biological functions of cell therapies. By embracing these principles and learning from both the successes and challenges of approved products, developers can build the compelling potency data packages needed to ensure consistent product quality, gain regulatory confidence, and ultimately deliver effective therapies to patients faster.

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