Navigating the Maze: A Strategic Comparison of EU and US CMC Requirements for Cell Therapies

Abstract

This article provides a comprehensive comparison of Chemistry, Manufacturing, and Controls (CMC) requirements for cell therapies in the European Union and United States. Aimed at researchers, scientists, and drug development professionals, it explores foundational regulatory frameworks, offers methodological guidance for global development, identifies common troubleshooting areas, and delivers a comparative validation analysis. The content synthesizes current guidelines, including the new EMA guideline on investigational ATMPs effective July 2025, to equip developers with actionable strategies for navigating transatlantic regulatory nuances and accelerating global market access.

Laying the Groundwork: Understanding EU and US Regulatory Frameworks for Cell Therapies

The development of advanced therapies represents one of the most innovative yet complex areas of modern medicine. These products, known as Advanced Therapy Medicinal Products (ATMPs) in the European Union (EU) and Cell and Gene Therapies (CGTs) in the United States, are subject to distinct regulatory frameworks that reflect different historical, legal, and scientific approaches to therapeutic oversight [1]. While both regulatory systems aim to ensure patient safety and product efficacy, their differing structures, definitions, and technical requirements create significant considerations for developers pursuing global approval pathways.

Understanding these nuances is crucial for researchers, scientists, and drug development professionals navigating the transition from laboratory research to clinical application. The regulatory landscape for these therapies is dynamic, with both the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) continuously updating their guidance to reflect scientific advancements and accumulated experience [2] [3]. This guide provides a detailed, objective comparison of the EU and US regulatory requirements, with a specific focus on Chemistry, Manufacturing, and Controls (CMC) aspects that are critical for successful product development and approval.

Definitions and Classification

The fundamental distinction between the regulatory systems begins with their classification approaches. The EU employs a more structured, legally defined categorization system, while the US utilizes a broader, more flexible umbrella terminology [1].

Table 1: Regulatory Classification Comparison

Aspect	European Union (ATMPs)	United States (CGTs)
Umbrella Term	Advanced Therapy Medicinal Products (ATMPs)	Cell and Gene Therapies (CGTs)
Sub-categories	- Gene Therapy Medicinal Product (GTMP)- Somatic Cell Therapy Medicinal Product (sCTMP)- Tissue Engineered Product (TEP)- Combined ATMP (cATMP)	- Human Gene Therapies- Somatic Cell Therapies
Key Differentiator	Precise legal delineation based on product nature and mechanism; new legislation (2023) includes genome editing and synthetic nucleic acids under GTMP [1].	Broader categorical nesting; no separate category for tissue-engineered products [1].
Combination Products	Products like CAR-T cells are always classified as Gene Therapy (GTMP) [1].	Genetically modified cells (e.g., CAR-T) are regulated by the Office of Therapeutic Products (OTP) under CBER [1].

The EU's classification system directly influences the specific regulatory pathway and data requirements. For instance, the Committee for Advanced Therapies (CAT) is the central committee responsible for ATMP classification and certification [1]. In the US, the Office of Therapeutic Products (OTP), within the Center for Biologics Evaluation and Research (CBER), regulates most CGTs [1]. Developers can seek formal classification through official procedures: a Request for Designation (RFD) with the FDA's Office of Combination Products, or a classification request to the EMA's CAT, both with a typical 60-day response time [1].

CMC and Manufacturing Requirements

Chemistry, Manufacturing, and Controls (CMC) represent a significant hurdle in advanced therapy development, with an estimated 74% of FDA Complete Response Letters (CRLs) from 2020-2024 citing manufacturing or quality deficiencies [4]. The regulatory expectations for CMC are phase-appropriate but rigorous in both regions, with particular emphasis on process control and analytical validation.

Starting and Raw Materials

The control of input materials is a critical area of regulatory divergence, impacting early development strategy and logistics.

Table 2: Comparison of Starting Material Requirements

Material Type	EMA (EU) Position	FDA (US) Position
General Definition	Materials that become part of the drug substance (e.g., vectors, cells) are "starting materials" and must be produced under GMP principles [2].	No formal regulatory definition of "starting materials"; uses "critical raw materials" with enhanced, risk-based control strategies [2].
Viral Vectors (for ex vivo modification)	Classified as a starting material. Once replication competent virus (RCV) is absent in the vector, the final cell product may not need further RCV testing [2].	Classified as a drug substance. Requires RCV testing on both the viral vector and the final cell-based drug product [2].
Donor Testing (Allogeneic)	Governed by the EU Tissue and Cell Directives (EUTCD); must be handled and tested in licensed, accredited premises [2].	Governed by 21 CFR 1271 Subpart C; expected to be tested in CLIA-accredited laboratories [2].

A notable difference lies in the acceptance of materials: the FDA does not allow research-grade excipients or starting materials for CGTs, even in early phases, due to potential patient risks [1]. The EMA also requires GMP-grade manufacturing for investigational products in first-in-human studies [1].

Process Validation and Control

As products advance to later stages, demonstrating a consistent and controlled manufacturing process becomes paramount. Both agencies expect increased process understanding and validation, though their specific expectations differ.

Table 3: Process Validation and Control Strategies

CMC Consideration	FDA (US) Position	EMA (EU) Position
Number of Validation Batches	Not explicitly specified; must be statistically adequate based on process variability [2].	Generally, three consecutive batches; some flexibility is allowed [2].
Use of Surrogate Approaches	Allowed, but must be thoroughly justified [2].	Allowed only in cases of a shortage in starting material [2].
Concurrent Validation	Allowed in certain circumstances [2].	Allowed for PRIME products and those addressing unmet medical needs [2].
Use of Platform Data	Acceptable where the same or similar manufacturing steps are used [2].	Acceptable where the same or similar manufacturing steps are used [2].

Analytical and Potency Testing

Demonstrating product quality, safety, and potency through robust analytical methods is a cornerstone of CMC. Both regulators apply a phase-appropriate approach, but expectations escalate significantly towards commercialization.

Analytical Methodologies

The FDA and EMA have shown openness to advanced and alternative analytical methods, provided they are scientifically justified [1].

• Orthogonal Methods: Both agencies encourage the use of orthogonal assays (methods using different scientific principles to measure the same attribute) to build confidence in critical quality attributes (CQAs) [1]. For example, identity, potency, and purity assays in gene therapy often require at least two complementary methods.
• Assay Validation: The FDA applies a phase-appropriate lens. Early-phase (IND) assays need to be qualified (reliable, reproducible, sensitive), while by Phase 3, full validation per ICH Q2(R2) is required [1]. The EMA's guideline for investigational ATMPs, effective July 2025, also states that orthogonal methods should be considered to ensure robustness, especially when standardized assays are lacking [1].
• New Approach Methodologies (NAMs): The FDA Modernization Act opened the door for in silico models or organ-on-chip technologies to supplement or replace certain in vivo studies, though acceptance is case-by-case and requires strong justification [1].

Potency Assay Requirements

Potency testing is frequently a major CMC challenge. The FDA expects functional, biologically relevant potency assays and identifies this as a common deficiency in CGT programs [1]. For viral vectors used in vitro, the FDA requires a validated functional potency assay for pivotal studies, whereas the EMA may find infectivity and transgene expression sufficient in early phases, with less functional assays sometimes acceptable later [2].

Table 4: Key Research Reagent Solutions for Analytical Development

Reagent / Technology	Primary Function in CGT/ATMP Analytics
Lentiviral & AAV Vector Systems	Serve as critical starting materials or drug substances for gene delivery; require full sequencing and RCV testing [5] [2].
qPCR & Digital PCR (dPCR)	Orthogonal methods for vector genome integrity and quantification, vector copy number determination, and detecting replication competent viruses [1] [6].
Next-Generation Sequencing (NGS)	Comprehensive profiling of vector genome integrity, identifying sequence variants, and ensuring genetic fidelity of final cell products [1].
Mass Photometry & Analytical Ultracentrifugation (AUC)	Advanced techniques for physical characterization, such as full/empty capsid ratio quantification and aggregation profiling for viral vector products [6].
Flow Cytometry & Immunophenotyping	Critical for cell therapy characterization, assessing identity, purity, transduction efficiency, and critical quality attributes of cellular products [7].

Demonstrating Comparability

Process changes are inevitable during development, necessitating comparability exercises. Currently, CGTs/ATMPs are outside the scope of ICH Q5E, though a new annex is in development [2]. In the interim, regional guidances apply.

• EU Approach: Relies on the EMA's 'Questions and Answers' document on ATMP comparability and multidisciplinary guidelines for genetically modified cells, which specify attributes to evaluate when changing the process for recombinant starting materials [2].
• US Approach: The FDA has issued draft guidance on CGT comparability (July 2023), reflecting current agency thinking [2].
• Shared Principles: Both agencies agree on a risk-based approach for the extent of testing, which should increase with the development stage. There is consensus on including stability testing and the usefulness of accelerated studies [2].
• Key Difference: The EMA guidance outlines specific tests for the finished product (e.g., transduction efficiency, vector copy number) when the starting material process changes. The FDA guidance currently lacks an equivalent specific provision [2]. Furthermore, while the FDA recommends including historical data in comparability exercises, the EMA does not require or recommend comparison to historical data [2].

Market Access and Post-Approval Considerations

Successfully navigating CMC requirements leads to market access challenges that differ substantially between the EU and US.

• EU Market Access Challenges: After a centralized EU marketing authorization, ATMPs must undergo country-by-country pricing and reimbursement negotiations, creating an "economic valley of death" [8]. A 2025 report indicated that of 19 authorized ATMPs, only 8 were marketed and reimbursed in Belgium, for example [8]. The new EU pharmaceutical legislation introduces Article 56a, which allows member states to require a marketing authorization holder to ensure sufficient product supply, which is challenging for personalized ATMPs with short shelf-lives [1].
• US Post-Approval Evolution: The FDA has demonstrated adaptability based on accumulated real-world evidence. In a significant 2025 shift, the FDA eliminated REMS requirements for CAR-T products, reducing the mandatory post-infusion monitoring period from four weeks to two and removing complex hospital certification rules [9]. This signals growing confidence in the safety management of these therapies and aims to improve patient access, especially in outpatient and community settings [9].
• Approval Statistics: As of 2025, the EU ATMP Regulation has produced 19 authorized ATMPs, with a clear dominance of GTMPs (16 products, 84.2%) over HCTPs like sCTMPs and TEPs (3 products) [8]. This highlights the significant regulatory and commercial hurdles for non-gene therapy ATMPs in Europe.

The regulatory landscapes for ATMPs in the EU and CGTs in the US, while converging on core scientific principles, present distinct pathways for developers. The EU's structured, legally precise classification system contrasts with the US's more flexible, umbrella-based approach. Key CMC differences are most pronounced in the classification of starting materials, expectations for potency assays, and specific comparability study requirements. Furthermore, the post-approval environment differs, with the EU facing fragmented market access and the US recently streamlining safety management requirements for established modalities like CAR-T.

For researchers and drug development professionals, these differences are not merely bureaucratic but have profound implications for CMC strategy, clinical development planning, and global market access. Success in this complex environment requires early and continuous engagement with regulators, a deep understanding of region-specific guidance, and the construction of robust, flexible manufacturing and control strategies that can meet the nuanced demands of both major markets.

For drug development professionals and researchers, navigating the regulatory pathways for cell therapies in the United States (US) and European Union (EU) presents significant challenges. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) serve as the principal regulatory bodies, but their organizational structures, technical requirements, and review processes differ substantially. Understanding these differences is crucial for successful global development strategies, particularly for Chemistry, Manufacturing, and Controls (CMC), which accounts for approximately 74% of complete response letters for cell and gene therapies according to recent FDA data [4].

This guide provides a detailed comparison of the FDA's Center for Biologics Evaluation and Research (CBER), specifically its Office of Therapeutic Products (OTP), and the EMA's Committee for Advanced Therapies (CAT). These bodies govern the approval of cell therapies—categorized as Cell and Gene Therapies (CGTs) in the US and Advanced Therapy Medicinal Products (ATMPs) in the EU [10] [11]. We objectively compare their structures, mandates, and the experimental data required for approval, providing a framework for researchers to navigate both regulatory landscapes efficiently.

Organizational Structures and Legal Mandates

US Framework: FDA/CBER/OTP

Within the FDA, CBER regulates cell and gene therapy products under the authority of the Public Health Service Act and the Federal Food, Drug, and Cosmetic Act [10]. The Office of Therapeutic Products (OTP) is the specific office under CBER responsible for reviewing these products [10] [12]. Its mandate encompasses the evaluation of CGTs from pre-investigational stages through to marketing approval and post-market surveillance. The OTP provides comprehensive oversight, reviewing preclinical data, clinical trial designs, CMC information, and facility readiness for Biologics License Applications (BLAs) [13].

EU Framework: EMA/CAT

In the EU, the Committee for Advanced Therapies (CAT) is a dedicated committee within the EMA that regulates ATMPs under Regulation (EC) No 1394/2007 [10] [1]. The CAT is responsible for assessing the quality, safety, and efficacy of ATMPs and preparing a scientific opinion for each marketing authorization application. The final approval decision is made by the European Commission based on the CAT's assessment [10]. The CAT also provides classifications for borderline products and offers scientific recommendations on the regulatory status of therapies [1].

Table 1: Key Structural and Legal Differences Between FDA/OTP and EMA/CAT

Aspect	FDA/CBER/OTP	EMA/CAT
Legal Authority	Public Health Service Act; FD&C Act [10]	Regulation (EC) No 1394/2007 [10]
Decision-Making Power	Full approval authority for CGTs [10]	Provides scientific opinion; European Commission grants final approval [10]
Product Scope	Cell and Gene Therapies (CGTs), including human gene therapy and cell therapy products [10] [11]	Advanced Therapy Medicinal Products (ATMPs): Gene Therapy, Somatic Cell Therapy, Tissue-Engineered Products, Combined ATMPs [10] [1]
Primary Guidance	FDA Guidance Documents (e.g., CMC guidance for CGTs) [12] [2]	EMA Guidelines (e.g., Guideline on clinical-stage ATMPs) [14] [2]

Diagram 1: Regulatory approval pathways for US and EU systems

Approval Pathways and Development Lifecycle

Clinical Trial Authorization

In the US, sponsors must submit an Investigational New Drug (IND) application to the FDA, including preclinical data, CMC information, and Institutional Review Board approval. The FDA has 30 days to review the application before clinical trials can begin, unless the study is placed on hold [10]. For the EU, a Clinical Trial Application (CTA) is submitted to National Competent Authorities and Ethics Committees. Since 2022, the centralized Clinical Trials Information System allows for submission across multiple EU states under the EU Clinical Trials Regulation [10]. The EU has set ambitious new targets to have two-thirds of clinical trials begin patient recruitment within 200 days of application submission [12].

Marketing Authorization and Expedited Pathways

For market approval in the US, a Biologics License Application must demonstrate safety, purity, and potency [10]. Standard review timelines are 10 months, or 6 months for priority review [10]. The EU requires a Marketing Authorization Application, with standard review taking 210 days, excluding clock stops, or 150 days for accelerated assessment [10].

Both regions offer expedited pathways, though with different structures. The FDA provides the Regenerative Medicine Advanced Therapy designation, Fast Track, Breakthrough Therapy, and Accelerated Approval [10] [12]. The EMA offers the PRIME scheme, Conditional Marketing Authorization, and Accelerated Assessment [10]. A critical difference lies in evidence requirements: the FDA often accepts real-world evidence and surrogate endpoints, particularly for rare diseases, while the EMA typically requires more comprehensive clinical data and longer patient follow-up [10].

Table 2: Comparison of Key Expedited Pathways and Approval Requirements

Parameter	FDA/CBER/OTP	EMA/CAT
Expedited Pathway	RMAT (Regenerative Medicine Advanced Therapy) [10] [12]	PRIME (Priority Medicines) [10]
Clinical Trial Review	30-day review for IND [10]	CTA review via National Competent Authorities & Ethics Committees [10]
Standard Review Timeline	10 months (standard), 6 months (priority) [10]	210 days (standard), 150 days (accelerated) [10]
Typical Evidence Requirements	More flexible; may accept surrogate endpoints, real-world evidence [10]	More comprehensive clinical data; larger patient populations; longer follow-up [10]
Post-Market Surveillance	Requires 15+ years LTFU for gene therapies; REMS for high-risk products [10]	Risk Management Plans; Periodic Safety Update Reports; EudraVigilance [10]

CMC Requirements: A Detailed Experimental Framework

Chemistry, Manufacturing, and Controls constitute a major area of regulatory divergence. Recent analysis shows CMC issues account for 74% of complete response letters for cell and gene therapies [4], emphasizing the critical need for robust manufacturing data.

Starting Materials and Raw Materials

The classification and control of starting materials differ significantly between regions. The EMA defines 'starting materials' as substances that become part of the drug substance, such as vectors or cells, requiring production under Good Manufacturing Practice principles [2]. In contrast, the FDA lacks a formal definition of starting materials, instead applying a risk-based approach to 'critical raw materials' with phase-appropriate control strategies [2].

For viral vectors used in modifying cell therapies, the FDA classifies them as a drug substance, while the EMA considers them starting materials [2]. This distinction has practical implications for potency assay requirements, with the FDA expecting more functional assays compared to the EMA's acceptance of infectivity and transgene expression assays, particularly in early development [2].

Demonstrating Comparability

Both agencies recognize that manufacturing process changes are inevitable during development, but their approaches to demonstrating comparability differ. While ICH Q5E currently excludes CGTs, both regions have issued specific guidance [2]. The FDA's draft guidance (2023) and the EMA's multidisciplinary guideline (effective July 2025) both advocate risk-based approaches, but differ in specific requirements [2].

For genetically modified cells, EMA guidance specifies exact attributes to assess when changing recombinant starting materials: full vector sequencing, absence of replication competent virus, impurity comparison, and stability assessment [2]. The FDA guidance lacks equivalent specificity but emphasizes comprehensive quality attribute assessment throughout development [2].

Diagram 2: Comparability assessment workflows for FDA and EMA

Process Validation and Control Strategies

Process validation requirements showcase both convergence and divergence. For the number of validation batches, the EMA typically requires three consecutive batches but allows flexibility, while the FDA does not specify a number but requires statistically adequate data based on process variability [2]. Both agencies allow use of platform data when similar manufacturing steps are used, and permit surrogate approaches in validation, though the EMA restricts this to cases of starting material shortage [2].

Regarding donor testing for cell therapies, the FDA follows 21 CFR 1271 Subpart C with testing expected in CLIA-accredited laboratories, while the EMA follows the European Union Tissues and Cells Directive, requiring handling and testing in licensed premises and accredited centres [2]. The EMA also requires some donor testing for autologous material, while the FDA's requirements differ [2].

Essential Research Reagents and Methodologies

Successful regulatory approval requires careful selection and validation of research reagents and analytical methods. The following toolkit outlines critical materials and their functions in CMC development.

Table 3: Research Reagent Solutions for Cell Therapy CMC Development

Reagent/Material	Function	Regulatory Considerations
GMP-Grade Viral Vectors	Gene delivery vehicle for genetically modified cell therapies	FDA: Drug substance [2]. EMA: Starting material [2].
Cell Separation Media	Isolation of target cell populations	Component affecting final product purity; requires qualification and traceability [1].
Cell Culture Media	Ex vivo expansion and maintenance of cells	Formulation changes may trigger comparability studies [2].
Critical Raw Materials	Ancillary materials used in manufacturing	Both agencies expect qualification and testing for identity, purity, and safety [1] [2].
Reference Standards	System suitability controls for analytical methods	Essential for assay validation; should be well-characterized and stored appropriately [14].
Functional Potency Assay Components	Measure biological activity of the product	FDA expects validated functional assays for pivotal studies [2].

Analytical Method Validation

Both regulators emphasize phase-appropriate assay validation. The FDA applies a phase-appropriate lens, requiring qualified methods for early-phase INDs that are reliable, reproducible, and sensitive enough for safety decisions, progressing to full validation under ICH Q2(R2) by Phase 3 [1]. The EMA's new clinical-stage ATMP guideline encourages orthogonal methods to ensure robustness when validated methods are lacking [1].

For potency assays—frequently cited as a CMC deficiency—the FDA expects biologically relevant functional assays rather than merely correlative measurements [1]. Both agencies show increasing openness to alternative methods, including New Approach Methodologies like in silico modeling and organ-on-chip systems, though sponsors must provide strong scientific justification for their use [1].

The regulatory landscapes of the FDA/CBER/OTP and EMA/CAT present both challenges and opportunities for cell therapy developers. The FDA's more centralized structure may enable streamlined decision-making, while the EMA's multi-cameral system provides specialized expertise through the CAT but requires coordination with the European Commission for final approval [10] [14].

For CMC strategies, developers should implement region-specific approaches for areas of significant divergence, such as viral vector classification and donor testing requirements, while leveraging harmonized approaches where principles align, such as risk-based comparability assessments [2]. Proactive regulatory engagement through FDA Type B meetings and EMA Scientific Advice is essential to anticipate differences early [10].

As global regulators push toward convergence through initiatives like the potential ICH Q5E annex for CGTs [2], developers must maintain agile CMC strategies. Understanding the detailed requirements and philosophical differences between these regulatory bodies will remain essential for efficiently advancing innovative cell therapies to patients in both markets.

In the global development of cell and gene therapies (CGTs), often classified as Advanced Therapy Medicinal Products (ATMPs) in the European Union, the classification and control of starting and raw materials present a critical and complex challenge [2] [15]. Early decisions regarding process, analytical, and manufacturing approaches directly impact licensure and commercialization, making a clear understanding of regulatory expectations paramount [2]. While the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) share the ultimate goal of ensuring patient safety and product quality, their regulatory frameworks for these materials contain nuanced differences in terminology, risk classification, and compliance expectations [2] [14] [15]. This guide objectively compares these requirements, providing a structured analysis for researchers, scientists, and drug development professionals navigating global CMC strategies.

Defining the Landscape: Raw vs. Starting Materials

A fundamental step in CMC strategy is the correct classification of materials used in the manufacturing process. The definitions and regulatory implications for these classifications diverge significantly between the EU and US, impacting control strategies and documentation.

Table: Comparative Definitions of Materials in the EU and US

Material Type	EMA / EU Regulatory Perspective	FDA / US Regulatory Perspective
Starting Material	A material that will become an integral part of the active substance (e.g., vectors for cell modification, gene editing components, cells) [2] [15].	No formal regulatory definition of "starting materials" [2].
Raw Material	Materials used during manufacture that are not intended to form part of the active substance (e.g., culture media, growth factors) [15].	Often uses the term "critical raw materials" to encompass most substances considered as starting or raw materials in the EU. Expects an enhanced, risk-based control approach [2].
Ancillary Material	Equates to the definition of raw materials [15].	A subset of raw materials that come into contact with the cell or tissue product but are not intended to be part of the final product [15].

Impact of Product Type on Material Classification

The classification of a material is not always absolute and can be influenced by the specific type of cell or gene therapy, requiring a case-by-case assessment [15].

• In Vivo Gene Therapy (e.g., Viral Vectors): For an adeno-associated viral (AAV) vector, the components used to create the vector—such as the master virus seed or the plasmids used to transfect the packaging cells and the master cell bank (MCB) of the packaging cell line—are typically considered starting materials, as they form the drug substance [15].
• Ex Vivo Gene Therapy (e.g., Genetically Modified Cells): For a therapy involving cells genetically modified with a lentiviral vector, the plasmids used to produce the lentivirus, the lentiviral vector itself, and the human cells are all considered starting materials [15]. In this complex case, a plasmid containing the transgene that becomes part of the drug substance is a starting material, while other supporting plasmids could be considered raw materials [15].

Table: Material Classification in Different Product Types

Therapy Type	Example Components	Typical Classification	Rationale
In Vivo Gene Therapy	Plasmids for vector production, Master Cell Bank	Starting Material	Forms an integral part of the Drug Substance [15].
Ex Vivo Gene Therapy	Lentiviral Vector, Human Cells	Starting Material	Components used to obtain the genetically modified cells [15].
General Manufacturing	Cell Culture Media, Growth Factors, Buffers	Raw Material	Supports manufacture but is not part of the final active substance [15].

Regulatory Consequences and Control Strategies

The classification of a material triggers specific requirements for quality control, Good Manufacturing Practice (GMP) compliance, and the level of detail required in regulatory submission dossiers [15]. Starting materials, being critical components, have a more direct impact on the Critical Quality Attributes (CQAs) of the final product and are therefore subject to more stringent controls [15].

Good Manufacturing Practice (GMP) Expectations

The point at which GMP requirements come into play for these materials differs between regions.

• European Union (EMA): The EMA has clear guidelines indicating that the donation, procurement, and testing of cellular starting materials can be performed outside of GMP [15]. However, as the process moves into the manufacture of the vector, cell purification, and the creation of master cell banks or viral seed stocks, full GMP control is expected [15]. A new GMP guide for ATMPs is also being proposed to allow flexibility in early development [15].
• United States (FDA): The FDA employs a more graduated, phase-appropriate approach to GMP compliance [14]. Early in clinical development, reliance on attestation is acceptable, with full GMP compliance verified during a pre-license inspection when a Biologics License Application (BLA) is submitted [14].

Practical Implications for Viral Vectors and Testing

The regulatory distinction for viral vectors used in ex vivo therapies is a prime example of divergent expectations. The FDA classifies in vitro viral vectors used to modify cell therapy products as a Drug Substance, whereas the EMA considers them to be Starting Materials [2]. This difference in classification cascades into testing requirements. For instance, for Replication Competent Virus (RCV) testing, the EMA may consider the requirement satisfied once absence is demonstrated on the in vitro vector. In contrast, the FDA requires that the cell-based drug product itself also be tested for RCV [2].

Essential Research Reagents and Materials

Successful navigation of the regulatory landscape for materials requires a robust quality system and a strategic approach to sourcing. The following table details key material categories and considerations for their use.

Table: Research Reagent Solutions for Cell and Gene Therapy Manufacturing

Material / Reagent Category	Key Examples	Critical Function in Manufacturing	Regulatory & Sourcing Considerations
Cellular Starting Materials	Patient/Donor Cells (Autologous/Allogeneic), Master Cell Banks	Forms the biological basis of the therapy; autologous vs. allogeneic determines process scale [16].	Donor screening and testing requirements differ by region. The FDA is more prescriptive than the EMA [2] [14].
Genetic Modifying Agents	Viral Vectors (Lentiviral, AAV), Plasmids, Gene Editing Components (mRNA, CRISPR-Cas9)	Introduces or modifies genetic material to impart the therapeutic mechanism of action [16].	Classification as a Starting Material (EU) or Drug Substance (US) affects GMP and testing requirements [2] [15].
Cell Culture & Expansion	Culture Media, Serum, Growth Factors, Cytokines, Buffers	Supports the ex vivo growth, survival, and differentiation of cells [15] [16].	"GMP grade" is not a certified term; implement a vendor qualification program. "Clinical grade" is a more reliable descriptor [15].
Process Reagents	Enzymes (e.g., Trypsin), Activation Agents, Transfection Reagents, Antibiotics	Enables specific manufacturing steps like cell detachment, activation, or genetic transfer [15].	For animal-derived components (e.g., porcine trypsin), specific guidelines exist for TSE/BSE risk mitigation [5] [16].
Analytical Reagents	Flow Cytometry Antibodies, ELISA Kits, PCR Master Mixes, Reference Standards	Used in identity, purity, potency, and safety assays to characterize the product and ensure quality [17] [16].	Methods must be phase-appropriately qualified/validated. Potency assays are critical for release [17] [18].

Experimental Protocols for Material Qualification

To ensure regulatory compliance, developers must establish rigorous experimental protocols for qualifying and controlling their materials. The following methodologies are foundational.

Protocol 1: Vendor and Raw Material Qualification

This workflow ensures that all raw materials are suitable for their intended use in the manufacture of a cell therapy product, aligning with both FDA and EMA expectations for a risk-based approach [2] [15].

Title: Raw Material Qualification Workflow

Methodology:

• Risk Assessment and Vendor Audit: Classify materials based on their criticality and risk to the process and product. Conduct a quality audit of the supplier to assess their quality system, manufacturing practices, and change control processes. Do not rely solely on "GMP grade" labels, as this is often a self-declared term [15].
• Define Specifications: Establish acceptance criteria for the material, including identity, purity, potency (if applicable), sterility, and endotoxin levels.
• Incoming Testing: Perform testing on received materials against the defined specifications. This may include certificates of analysis review and identity confirmation [16].
• Feasibility and Compatibility Study: Test the material in a small-scale model of the manufacturing process to ensure it performs as expected and does not adversely impact cell growth, function, or final product CQAs.
• Documentation and Change Control: All qualification data must be documented in the quality system. A robust change control process must be established to manage any changes from the vendor or to an alternative source [17].

Protocol 2: Plasmid DNA Suitability for Viral Vector Production

This protocol outlines the characterization of plasmid DNA, a common starting material for viral vectors, to ensure it is fit for purpose [15].

Methodology:

• Sequence Verification: Perform full plasmid sequencing to confirm the identity and correctness of the genetic elements (e.g., transgene, promoter, resistance gene) [2].
• Purity and Impurity Analysis: Use orthogonal methods like agarose gel electrophoresis to assess supercoiled vs. linear conformations and HPLC-SEC to quantify product-related impurities and dimers [17].
• Sterility and Mycoplasma Testing: Conduct compendial sterility and mycoplasma tests to ensure the plasmid is free from microbial contamination [16].
• Functionality Testing: Use the plasmid in a small-scale vector production run. Assess critical quality attributes of the resulting vector, such as infectivity, vector copy number, and transgene expression, to confirm the plasmid's functionality [2]. This is a key expectation in EMA guidance for recombinant starting materials [2].

Regulatory Guidance and Future Directions

Navigating the requirements for starting and raw materials requires a firm grounding in the available regulatory guidance documents.

• European Medicines Agency (EMA): The primary source is the "Guideline on human cell-based medicinal products" (EMEA/CHMP/410869/2006) and the multidisciplinary "Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials" effective July 2025 [5] [14]. The European Pharmacopoeia chapter 5.2.12 is also relevant for raw materials of biological origin [15].
• US Food and Drug Administration (FDA): Key guidance includes "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)" (January 2020) and the recent draft "Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products" (July 2023) [19].

A significant trend is the pursuit of global regulatory convergence to streamline development. Initiatives like the FDA's Gene Therapies Global Pilot Program (CoGenT) aim to foster collaborative reviews with international partners like the EMA [20]. Furthermore, a new Annex to the ICH Q5E guideline on comparability is in development, which will address CGT-specific challenges and may further harmonize expectations for materials and process changes [2].

The definitions and regulatory expectations for starting and raw materials in cell and gene therapy represent a tale of two regions, with the EU employing more defined terminology and the US adopting a risk-based, phase-appropriate approach. These differences directly impact GMP implementation, testing strategies, and regulatory submission content [2] [14] [15]. Success in global development hinges on a deep, case-by-case understanding of these nuances, early engagement with regulators via pre-IND meetings, and the implementation of a robust, risk-based quality system for material qualification and control [2] [16]. As the regulatory landscape evolves toward greater convergence, a proactive and strategic approach to CMC planning for materials remains a critical determinant of speed to market and patient access.

The European Union is undergoing its most significant pharmaceutical legislative reform in over two decades, creating a new operational reality for developers of cell and gene therapies [21] [22]. For researchers and scientists navigating the complex Chemistry, Manufacturing, and Controls (CMC) requirements for Cell Therapy Products (CTPs), understanding the interplay between the new Pharma Package and the Substances of Human Origin (SoHO) Regulation is critical. These regulations, set to take effect in the coming years, collectively aim to ensure patient safety, improve supply security, and support innovation, yet they also introduce new compliance layers for CTP development [23] [24].

Framed within a broader thesis comparing EU and US cell therapy CMC requirements, this guide objectively analyzes how these legislative changes impact manufacturing strategies, analytical methods, and regulatory planning. The evolving EU framework demonstrates both divergence from and alignment with US Food and Drug Administration (FDA) approaches, particularly in the classification of starting materials, environmental monitoring, and supply chain obligations [2] [1]. This analysis provides drug development professionals with the experimental protocols and comparative data necessary to build robust, cross-compliant development strategies in this dynamic regulatory environment.

Comparative Analysis: Key Legislative Changes and Their CMC Implications

The EU Pharma Package: Incentives and Obligations

The Pharma Package, the first major revision since 2004, aims to balance enhanced incentives for innovation with strengthened obligations for access and supply [21] [25]. Key elements impacting CTP development include modified exclusivity periods, new supply requirements, and incentives for unmet medical needs.

Table 1: Key Elements of the EU Pharma Package Impacting Cell Therapies

Component	Key Provision	Impact on Cell Therapy Development
Regulatory Data Protection	8 years of data protection [21] [22]	Protects innovative CTPs from generic competition based on submitted data.
Market Protection	1 year of market protection, extendable to 2 years [21] [22]	Grants de facto market exclusivity; extensions possible for addressing unmet needs.
Orphan Medicinal Products	Market exclusivity of 10-12 years possible [21] [1]	Significant incentive for rare disease CTP development.
Transferable Exclusivity Vouchers	Vouchers for new antimicrobials, usable in 5th year of RDP with revenue cap [25] [22]	Less directly relevant to CTPs, but indicates innovative incentive mechanisms.
Obligation to Supply (Article 56a)	Member States can require MAH to supply sufficient quantities [26] [22]	Challenging for personalized CTPs with complex logistics; may require local manufacturing.

The SoHO Regulation: Quality and Safety for Starting Materials

The SoHO Regulation (EU) 2024/1938, applying fully from 7 August 2027, establishes stringent standards for quality and safety for all substances of human origin intended for human application [27] [24]. This directly affects CTPs, which often rely on human cells as starting materials. The regulation extends activities covered to include donor registration, collection, testing, storage, distribution, import, and export [1]. Any entity handling these activities, now termed "SoHO entities," must comply, adding a layer of regulatory oversight for cell therapy developers [1]. The primary goal is to protect human health by bolstering the continuity of supply for critical substances while ensuring robust donor and recipient protection [23].

Comparative Workflow: EU vs. US Cell Therapy Regulatory Pathways

The following diagram illustrates the parallel yet distinct regulatory pathways for a Cell Therapy Product in the EU and US, highlighting key stages where the new Pharma Package and SoHO Regulation introduce specific requirements.

Experimental Protocols and CMC Requirements: A Side-by-Side Comparison

Navigating the CMC requirements for cell therapies requires a detailed understanding of where EU and US regulations converge and diverge. The following experimental protocols and data are critical for a successful global development strategy.

Protocol 1: Starting Material Definition and Qualification

The classification and quality standards for starting materials represent a fundamental difference between EU and US regulators, directly impacting initial process design.

Table 2: Comparison of Starting Material Requirements for a Genetically Modified Cell Therapy

Parameter	EMA/SoHO Position	FDA Position	Experimental Consideration
Viral Vector for ex vivo modification	Considered a Starting Material [2]. Must be produced under GMP principles; site inspections are uncommon but quality must be assured by the Qualified Person (QP) [2].	Classified as a Drug Substance [2]. Requires facility licensing and inspection for quality metrics (purity, potency, safety) [11].	EU: Focus on full traceability and GMP for vector manufacturing. US: Plan for BLA and pre-license inspection of the vector manufacturing facility.
Donor Testing (Autologous)	Governed by EUTCD. Requires some donor testing even for autologous material [2]. Testing must be handled in licensed premises and accredited centres [2].	Governed by 21 CFR 1271 Subpart C. Expected to be tested in CLIA-accredited labs [2].	EU: Ensure all donor testing labs are accredited under EU standards. US: CLIA certification is a key prerequisite for clinical labs.
Replication Competent Virus (RCV) Testing	Once absence is demonstrated on the viral vector, the resulting genetically modified cells do not typically require further RCV testing [2].	Requires that the final cell-based drug product also needs to be tested for RCV [2].	EU: Testing strategy can focus on the vector. US: Budget for additional, validated RCV testing on the final drug product lot.

Protocol 2: Demonstrating Manufacturing Process Comparability

Both agencies recognize that process changes are inevitable during development. However, the analytical burden for demonstrating comparability differs.

Table 3: Comparability Exercise Design for a Major Process Change

Analytical Method	EMA Expectations	FDA Expectations	Key Analytical Nuances
Potency Assay	Infectivity and transgene expression may suffice in early phase; less functional assays can be acceptable later [2].	A validated, functional potency assay is essential to assess the efficacy of the drug product used in pivotal studies [2].	US: Invest early in developing a biologically relevant functional assay (e.g., based on mechanism of action).
Stability Data	Real-time data is not always needed to support a comparability conclusion [2].	A thorough assessment, including real-time data for certain changes, is expected [2].	US: Plan for side-by-side real-time stability studies to support any major process change before Phase 3.
Use of Historical Data	Comparison to historical data is not required/recommended [2].	The inclusion of historical data is recommended to establish a baseline [2].	US: Maintain extensive in-house historical databases for critical quality attributes (CQAs).
Attribute-Specific Testing (for GM cells)	Specific tests for finished product are required (e.g., transduction efficiency, vector copy number, transgene expression) when changing recombinant starting materials [2].	No direct equivalent in the current FDA guidance for starting material changes [2].	EU: A defined set of quality attributes must be monitored for genetically modified cell products.

Protocol 3: Process Validation and Commercial Readiness

As programs advance towards commercialization, validation strategies must account for regional expectations, particularly given the small batch sizes and inherent variability of CTPs.

Table 4: Process Validation and Commercialization Requirements

CMC Consideration	FDA Position	EMA Position	Commercialization Strategy
Number of Validation Batches	Not specified, but must be statistically adequate based on process variability [2].	Generally, three consecutive batches. Some flexibility is allowed [2].	EU: The "three-batch" paradigm is a standard expectation. US: Justify the number of batches with statistical power analysis.
Use of Surrogate Approaches	Allowed, but must be thoroughly justified [2].	Allowed only in case of a shortage in starting material (e.g., limited patient cells) [2].	EU: Surrogates are not a default strategy; use only with a robust justification tied to material scarcity.
Concurrent Validation	Allowed in certain circumstances [2].	Allowed for PRIME products and those addressing unmet medical needs [2].	EU: Expedited pathways may offer more flexible validation options.
Use of Platform Data	Acceptable where the same or similar manufacturing steps are used across multiple products [2].	Acceptable where the same or similar manufacturing steps are used [2].	Harmonized: Both agencies are open to platform validation approaches for standardized processes.

The Scientist's Toolkit: Essential Research Reagent Solutions

The following reagents and materials are critical for conducting the CMC analytical work described in the experimental protocols above, particularly for meeting the quality and comparability standards of both EU and US regulators.

Table 5: Key Research Reagent Solutions for Cell Therapy CMC Development

Reagent/Material	Function in CMC Development	Regulatory Application & Consideration
GMP-Grade Viral Vectors	Used as starting material or drug substance for genetic modification of cells.	EU: Must be produced under GMP as a starting material [2]. US: Classified as a drug substance, requiring stringent control [2].
Reference Standard & Panel Cells	Critical for assay qualification/validation, particularly for potency and identity testing.	Required by both agencies. The panel must be well-characterized to establish assay suitability and range.
Functional Potency Assay Kits	Measure biologically relevant activity of the cell product (e.g., cytotoxic activity, differentiation potential).	US: Emphasis on a validated functional assay for BLA [2]. EU: Moving towards stronger functional assay requirements.
Vector Copy Number (VCN) Assays	Quantify the number of integrated vector genomes per cell, a key safety and consistency attribute.	Critical CQAs for genetically modified cells in both regions, especially for EU comparability exercises [2].
Cell Sorting Reagents (e.g., Antibodies, Beads)	Isulate specific cell populations to ensure product purity and identity.	Purity and identity are universal CQAs. Reagents must be qualified and their impact on the final product understood.
Mycoplasma Detection Kits	Essential for sterility testing and ensuring freedom from mycoplasma contamination.	Mandatory safety test for both EMA and FDA. The method must be validated according to pharmacopoeial standards.

The concurrent implementation of the EU Pharma Package and SoHO Regulation creates a more complex but potentially more predictable environment for cell therapy developers. The strategic implications are profound: successful global development programs will be those that integrate regulatory strategy with CMC planning from the earliest stages.

The new EU framework, with its emphasis on supply chain obligations, necessitates early decisions about manufacturing network design, especially for personalized therapies [26] [1]. Conversely, the extended incentives for orphan drugs present a significant opportunity for rare disease CTPs [21] [25]. The divergence from US requirements on starting materials and comparability means that a one-size-fits-all CMC strategy is not viable; instead, developers must create a core global data package with region-specific modules [2] [1].

For researchers and scientists, proactive engagement is key. Utilizing early regulatory advice mechanisms like the EMA's Committee for Advanced Therapies (CAT) classification and the FDA's INTERACT meetings can de-risk development and clarify regional expectations [1]. By understanding these evolving legislative landscapes and their direct impact on CMC requirements, drug development professionals can better navigate the path to market, ensuring that innovative cell therapies reach patients in both the EU and US efficiently and safely.

For developers of cell and gene therapies, known as Advanced Therapy Medicinal Products (ATMPs) in the European Union and Cell and Gene Therapies (CGTs) in the United States, early and strategic engagement with regulators is a critical success factor [1] [11]. The complexity of these products, coupled with evolving regulatory frameworks, makes it essential to seek early feedback to de-risk development and align programs with regional expectations. This guide provides a detailed comparison of three pivotal early-stage regulatory interactions: the FDA's INTERACT and Pre-IND meetings in the US, and the CAT Classification procedure in the EU.

Understanding the distinct purpose, timing, and output of each pathway enables sponsors to build a more robust Chemistry, Manufacturing, and Controls (CMC) strategy from the outset, which is vital for global development plans [2].

US FDA Early-Stage Meetings

The US Food and Drug Administration (FDA) offers structured, non-binding meetings to guide sponsors through the initial phases of product development. The choice between an INTERACT and a Pre-IND meeting depends on the maturity of the development program and the specific nature of the questions.

INTERACT Meeting

The Initial Targeted Engagement for Regulatory Advice (INTERACT) meeting is an informal, early consultation mechanism for novel products that present unique challenges related to safety profiles, complex manufacturing, or innovative technologies [28] [29].

• Purpose and Focus: To obtain initial, non-binding advice on CMC, pharmacology/toxicology, and early clinical aspects before a definitive development path is established [29] [30]. It is ideal for addressing novel challenges and identifying potential roadblocks.
• Timing: Requested once the investigational product has been identified and some preliminary proof-of-concept studies are completed, but before definitive toxicology studies are designed or conducted, and before the clinical manufacturing process is finalized [28] [29].
• Key Outcomes: The feedback serves as a directional compass, helping to shape the initial development plan, understand the regulatory pathway, and avoid costly missteps later on. The FDA does not generate meeting minutes, though sponsors can submit their own for comment [30].

Pre-IND Meeting

The Pre-Investigational New Drug (Pre-IND) meeting is a more structured interaction designed to discuss specific plans for an imminent IND application [29].

• Purpose and Focus: To review and get feedback on the design of preclinical studies, the initial clinical study protocol, and the product manufacturing and quality controls needed to initiate human trials [29]. The focus is on the adequacy of the IND-enabling data package.
• Timing: Appropriate when the sponsor has a more advanced program, including a defined manufacturing process for clinical studies, developed assays with preliminary release criteria, and completed proof-of-concept studies, potentially including some preliminary safety/toxicology studies [29].
• Key Outcomes: More detailed feedback on the proposed IND content, which helps to prevent clinical holds and ensures the application is complete and adequate for review.

Strategic Workflow for US FDA Early Engagement

The following diagram illustrates the decision-making process and key milestones for selecting and preparing for an INTERACT or Pre-IND meeting.

EU CAT Classification Advice

In the European Union, the Committee for Advanced Therapies (CAT) is the central committee responsible for assessing the quality, safety, and efficacy of ATMPs. A fundamental first step for any developer is to determine how their product is classified.

• Purpose and Focus: The CAT classification procedure determines whether a product qualifies as a gene therapy, cell therapy, tissue-engineered product, or combined ATMP [1]. This classification is critical as it dictates the specific regulatory requirements for development and marketing authorization. For example, a product that is a combination of a cell and gene therapy, such as CAR-T cells, is always classified as a gene therapy in the EU [1].
• Timing: It is advisable to seek classification early in development, before embarking on a significant clinical program, to ensure the correct regulatory pathway is followed from the start [1].
• The Process: Sponsors submit a request for classification directly to the CAT using a specific form. The CAT aims to respond within 60 days [1]. This formal opinion on the product's legal status is a distinctive feature of the EU regulatory system.

Comparative Analysis: INTERACT, Pre-IND, and CAT Classification

The following table provides a side-by-side comparison of the three early-stage engagement mechanisms, highlighting their key characteristics and requirements.

Table 1: Comparative Analysis of Early-Stage Regulatory Engagement Pathways

Feature	FDA INTERACT Meeting	FDA Pre-IND Meeting	EU CAT Classification
Primary Goal	Obtain initial, non-binding advice on novel challenges [29]	Get detailed feedback on IND-enabling studies & clinical plans [29]	Obtain a formal opinion on product legal classification [1]
Ideal Timing	After preliminary PoC, before definitive toxicology & final CMC [28] [29]	After PoC, with defined CMC process, before IND submission [29]	Early in development, before major investment in clinical program [1]
Development Phase	Pre-pre-IND, very early development	Late pre-clinical, immediately pre-IND	Pre-clinical or early clinical
Key Questions	"Does the FDA agree with our overall development concept?"	"Is our pre-clinical and CMC data package sufficient for the IND?"	"What is the legal classification of our product?"
Formality & Output	Informal; no binding advice or formal FDA minutes [30]	Formal meeting; written FDA feedback is provided	Formal procedure; written classification opinion is provided
Official Timeline	Meeting held within 90 days of request [30]	Varies, but scheduled per FDA guidance	Response within 60 days of request [1]

Essential Research Reagents and Materials for Early CMC Development

A successful early-stage regulatory interaction, particularly concerning CMC, requires supporting data generated with well-characterized materials. The table below details key reagents and their functions in building the initial CMC package.

Table 2: Key Research Reagent Solutions for Early-Stage CMC Development

Reagent/Material	Function in CMC Development	Relevant Regulatory Context
Viral Vectors (e.g., AAV, Lentivirus)	Used as a delivery vehicle for gene therapies or to genetically modify cells (e.g., CAR-T). Critical for evaluating transduction efficiency and safety [2].	In the US, often classified as a drug substance [2]. In the EU, typically considered a starting material, but must be produced under GMP principles [2] [11].
Cell Lines & Primary Cells (Autologous/Allogeneic)	The core active component of cell-based therapies. Used to establish and optimize expansion, differentiation, and genetic modification processes [2].	Donor testing requirements differ between FDA (21 CFR 1271) and EMA (EUTCD). EMA may require some testing even for autologous material [2].
Gene Editing Components (e.g., CRISPR-Cas9)	The active agents for in-situ gene editing. Require thorough characterization of editing efficiency and specificity (on-target vs. off-target) [1].	EMA's guidelines define ex vivo genome editing machinery as starting materials [1].
Cell Culture Media & Supplements	Supports the growth and maintenance of cells during manufacturing. The quality and consistency directly impact product quality and safety [2].	Regarded as critical raw materials. Quality must increase with development phase, moving away from research-grade to fully qualified, GMP-compliant materials [1].
Functional Potency Assays	Analytical methods designed to measure the biological activity of the product, which is directly linked to its intended mechanism of action [2].	A common CMC deficiency. The FDA expects validated functional potency assays for pivotal studies. EMA may accept infectivity and transgene expression earlier, but expects more functional assays later [2].

Navigating the early regulatory landscape for cell and gene therapies requires a strategic and informed approach. The choice between an INTERACT and a Pre-IND meeting hinges on the maturity of your CMC and non-clinical data, while engaging with the EU CAT for classification is a foundational step for any European development program.

By understanding the distinct purposes, optimal timing, and expected outcomes of these pathways, developers can build a more robust global strategy. Leveraging these early dialogues, supported by data from well-characterized reagents and materials, significantly de-risks development, helps avoid costly delays, and paves the way for successful market authorization in both the US and EU.

Building Your CMC Strategy: Practical Approaches for EU and US Compliance

For developers of cell and gene therapies (CGTs), Chemistry, Manufacturing, and Controls (CMC) activities represent a critical pathway where early decisions directly impact successful licensure and commercialization. A phase-appropriate CMC strategy provides a structured framework that balances regulatory compliance with practical development needs, evolving from fit-for-purpose methods in early development to fully validated processes for commercial application [31] [32]. This approach is particularly crucial for companies pursuing global development programs that must satisfy both U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) requirements [2].

The fundamental principle of phase-appropriate CMC development acknowledges that the level of product and process understanding deepens over time. Initially, the focus is on ensuring patient safety and supporting proof-of-concept studies. As products advance through clinical development, the emphasis shifts toward rigorous process characterization, validation, and establishing comprehensive specifications to ensure consistent product quality [32]. Navigating the nuanced differences between FDA and EMA expectations for this progression is essential for efficient global development, potentially reducing costly delays and facilitating faster patient access to transformative therapies [2] [14].

Comparative Analysis of EU and US Regulatory Frameworks for CMC Development

Foundational Guidelines and Regulatory Bodies

The regulatory landscapes for advanced therapies in the EU and US are governed by distinct frameworks and agencies. In the US, the FDA's Center for Biologics Evaluation and Research (CBER), specifically its Office of Therapeutic Products (OTP), oversees cell and gene therapy products [19] [33]. The FDA has issued numerous product-specific and general guidances covering CMC, non-clinical, and clinical aspects [19]. In the EU, the EMA's Committee for Advanced Therapies (CAT) is responsible for evaluating advanced therapy medicinal products (ATMPs) [5]. A significant recent development is the EMA's new multidisciplinary guideline on investigational ATMPs, which came into effect in July 2025 and consolidates recommendations from over 40 previous documents [14].

Table 1: Key Regulatory Guidance Documents for Cell Therapy CMC Development

Development Phase	US FDA Guidance	EU EMA Guidance
Early Clinical	Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) (2020) [19]	Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products (2025) [14]
CMC & Manufacturing	Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products (Draft, 2023) [19]	Questions and answers on comparability considerations for advanced therapy medicinal products (ATMP) (2019) [5]
Potency Testing	Potency Assurance for Cellular and Gene Therapy Products (Draft, 2023) [19]	Guideline on potency testing of cell based immunotherapy medicinal products for the treatment of cancer (2006) [5]
Late-Stage & Commercial	Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products (2024) [19]	Guideline on human cell-based medicinal products (2006) [5]

Key Regulatory Divergence in CMC Requirements

While many core CMC principles align between regions, several critical differences impact strategic development planning. Understanding these nuances enables sponsors to design more efficient global CMC programs.

Starting Materials and Raw Materials: The EMA explicitly defines 'starting materials' as materials that become part of the drug substance, such as vectors and cells, requiring them to be produced under Good Manufacturing Practice (GMP) principles. In contrast, the FDA uses the term 'critical raw materials' and expects a risk-based control approach appropriate to the development stage [2]. This distinction is particularly relevant for viral vectors used to modify cell therapy products, which the FDA classifies as a drug substance, while the EMA considers them starting materials [2].

Donor Testing Requirements: For allogeneic therapies using human-derived materials, the FDA provides prescriptive requirements for donor eligibility determination, including specified testing for communicable diseases in qualified laboratories. The EMA also requires compliance with the European Union Tissues and Cells Directive (EUTCD) but references member state-specific legal requirements, creating a more fragmented landscape that requires case-by-case analysis [2] [14].

Process Validation and Comparability: The EMA typically expects three consecutive batches for process validation, with limited flexibility. The FDA does not specify a fixed batch number, instead requiring a "statistically adequate" number based on process variability [2]. For demonstrating comparability after process changes, the EMA's guidance for genetically modified cells outlines specific attributes to evaluate for both the starting material and finished product. The FDA's draft guidance on comparability (2023) reflects a more general risk-based approach without equivalent specific test recommendations [2].

Table 2: Comparative Analysis of Key CMC Requirements: FDA vs. EMA

CMC Aspect	US FDA Position	EU EMA Position
Starting Materials	Risk-based 'critical raw material' approach; viral vectors = drug substance [2]	GMP for 'starting materials'; viral vectors = starting material [2]
Donor Testing	Governed by 21 CFR 1271; tested in CLIA-accredited labs [2]	Governed by EUTCD; handled in licensed premises [2]
Process Validation Batches	Statistically adequate number [2]	Generally three consecutive batches [2]
Use of Surrogate Data in PV	Allowed with justification [2]	Allowed only with starting material shortage [2]
Stability Data for Comparability	Thorough assessment with real-time data for certain changes [2]	Real-time data not always required [2]
GMP Compliance Emphasis	Phase-appropriate approach; verification at BLA [14]	Mandatory GMP compliance for clinical trials [14]

Phase-Appropriate CMC Development: From Preclinical to Commercial

Analytical Method Life Cycle Management

The evolution of analytical methods parallels the product development lifecycle, transitioning from fit-for-purpose to fully validated status. This progression ensures that methods generate reliable data to support critical decisions at each phase while efficiently utilizing resources [32].

Preclinical to Early Clinical Phase (Phase 1): The primary focus is identifying potential Critical Quality Attributes (CQAs) and developing methods to measure them. According to ICH Q8(R2) and ICH Q11 principles, sponsors should create a Target Product Profile (TPP), define a quality TPP, and establish CQAs through risk assessment [32]. At this stage, method validation is typically not required for an original IND or Investigational Medicinal Product Dossier (IMPD). However, sponsors must demonstrate that test methods are appropriately controlled and scientifically sound [32]. For cell therapies, assays critical to patient safety, such as those determining dose or testing for replication-competent vectors, should be qualified before clinical studies begin [32].

Late Clinical Phase (Phase 2-3): As development advances toward pivotal trials, CQAs are refined based on accumulated process and product knowledge. Method qualification activities demonstrate that methods are fit-for-purpose and perform in line with the established Analytical Target Profile (ATP) [32]. The ATP is a prospective, technology-independent description of the desired performance of an analytical procedure, defining the required quality of the reportable value and serving as a basis for qualification criteria [32].

Commercial Phase (BLA/MAA): Prior to marketing application submission, analytical methods must be fully validated according to ICH Q2(R2) guidelines [32]. The method lifecycle continues post-approval with ongoing performance monitoring to ensure continued suitability throughout the product's commercial life.

Process Validation and Control Strategies

A phase-appropriate approach to process validation acknowledges that process understanding deepens throughout development. Early-phase manufacturing focuses on producing material with sufficient quality to support safety assessments, while late-phase activities emphasize process robustness, characterization, and validation to ensure consistent commercial manufacturing [2] [34].

The FDA allows greater use of platform data and surrogate approaches in process validation when justified, while the EMA accepts platform approaches only where similar manufacturing steps are used and restricts surrogate approaches to cases of starting material shortage [2]. For CGT products with accelerated approval pathways, such as those with RMAT designation in the US, CMC development timelines may be compressed, requiring closer alignment between clinical and CMC activities [35]. The FDA notes that regenerative medicine therapies with expedited clinical development may "face unique challenges in expediting product development activities to align with faster clinical timelines" [35].

Experimental Protocols and Methodologies for Critical CMC Activities

Analytical Method Bridging Studies

When introducing new or revised analytical methods with improved robustness, sensitivity, or operational simplicity, a bridging study is required to demonstrate comparability with the existing method. According to ICH Q14 guidelines on analytical procedure development, the design and extent of bridging studies should establish the numerical relationship between the reportable values of each method and assess the impact on product specification [32].

Protocol Overview:

• Study Design: The bridging strategy should be risk-based and consider the product development stage, ongoing studies, and the number of retained historical batches.
• Sample Analysis: Test a sufficient number of samples (typically 6-12) representing expected product variability using both the current and new methods.
• Data Analysis: Apply statistical methods (e.g., linear regression, equivalence testing) to establish correlation between methods.
• Acceptance Criteria: Define pre-established criteria for method comparability based on the method's intended use and its impact on quality decisions.

Potency Assay Development

Potency assays represent some of the most complex analytical methods to develop and validate for CGT products. These quantitative assays measure the biological response elicited by a product and are required components of quality, batch release, and stability testing [31].

Experimental Workflow:

• Mechanism of Action (MOA) Analysis: Identify the biological activity most relevant to the product's therapeutic effect.
• Assay Platform Selection: Choose appropriate platforms based on MOA, which may include cell-based assays, receptor/ligand binding assays, flow cytometry, or molecular methods like qPCR/dPCR [31].
• Assay Development and Optimization: Define critical assay parameters through design of experiments (DoE) approaches.
• Method Qualification/Validation: Establish assay precision, accuracy, linearity, range, and robustness according to phase-appropriate requirements.

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Research Reagents and Materials for Cell Therapy CMC Development

Reagent/Material	Function	CMC Application
Cell Culture Media	Supports cell growth, expansion, and maintenance	Manufacturing process development; critical raw material requiring qualification [2]
Viral Vectors	Gene delivery vehicles for genetic modification	Starting material (EMA) or drug substance (FDA); requires functional potency assays [2]
Cytokines/Growth Factors	Direct cell differentiation and functionality	Critical process reagents; impact product quality and potency [31]
Flow Cytometry Antibodies	Cell phenotype and characterization analysis	Identity, purity, and potency testing; method development for CQA assessment [31] [32]
qPCR/dPCR Reagents	Genetic analysis and vector copy number determination	Quality control and safety testing; critical for characterizing genetically modified cells [31] [32]
Reference Standards	Analytical method calibration and system suitability	Potency assay qualification; essential for method validation and comparability [32]

Strategic Implementation of Global CMC Development Programs

Harmonization Opportunities and Challenges

Despite regulatory differences, there are significant opportunities for harmonization in global CMC development. Both FDA and EMA emphasize risk-based approaches to evaluating quality attributes and agree that the extent of testing should increase with the development stage [2]. Both agencies recognize that accelerated or stress studies can identify differences in stability-indicating attributes, though they may differ on when real-time stability data is required [2].

The recent EMA guideline on clinical-stage ATMPs demonstrates substantial regulatory convergence in CMC content organization, which largely mirrors Common Technical Document (CTD) section headings familiar to FDA submissions [14]. However, terminology differences persist—EMA uses "Active substance" and "Investigational medicinal product" versus FDA's "Drug substance" and "Drug product" [14].

Accelerated Development Pathways

Both regions offer expedited programs for regenerative medicine therapies addressing serious conditions. The FDA's Regenerative Medicine Advanced Therapy (RMAT) designation and the EMA's Priority Medicines (PRIME) scheme provide opportunities for enhanced regulatory interaction and accelerated assessment [35]. However, these expedited clinical pathways create CMC challenges, as sponsors must accelerate product development activities to align with faster clinical timelines [35].

For sponsors using accelerated pathways, FDA recommends early engagement with the Office of Therapeutic Products (OTP) to discuss CMC development plans [35]. Importantly, expedited designation does not reduce CMC information requirements for assuring product quality. If manufacturing changes occur post-designation, the product may no longer qualify if comparability cannot be established [35].

Successful global development of cell therapies requires a sophisticated understanding of both FDA and EMA CMC requirements implemented through a phase-appropriate strategy. While regulatory convergence continues to evolve, key differences in starting material classification, donor testing, process validation, and analytical method expectations necessitate careful planning from the earliest development stages.

The progression from fit-for-purpose to validated processes represents a journey of increasing product and process understanding, mirrored by more stringent regulatory expectations. By identifying critical divergence points early and building flexible CMC strategies that accommodate both regulatory frameworks, sponsors can optimize their development programs for global success, ultimately accelerating patient access to transformative cell and gene therapies.

In the development of cell and gene therapies (CGTs), known as Advanced Therapy Medicinal Products (ATMPs) in the European Union, a robust control strategy is paramount for regulatory success. This strategy ensures that critical quality attributes (CQAs) of the final product are consistently met by identifying and controlling critical process parameters (CPPs) throughout manufacturing. A risk-based approach to linking CPPs to CQAs is not merely a regulatory expectation but a fundamental component of Chemistry, Manufacturing, and Controls (CMC) planning that aligns with both the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) frameworks [2]. The complexity and inherent variability of biological systems, particularly for autologous therapies, make this approach essential for demonstrating product consistency, especially when navigating the nuanced differences between major regulatory jurisdictions [1].

Analysis of recent FDA Complete Response Letters (CRLs) reveals that a significant 74% of applications were rejected for manufacturing or quality issues, underscoring the critical importance of a well-defined control strategy [4]. This guide provides a comparative analysis of experimental methodologies for developing these strategies within the context of divergent EU and US regulatory landscapes, offering researchers a structured pathway for global development.

Comparative Regulatory Landscape: US vs. EU Fundamentals

Foundational Principles and Terminology

While both regulators endorse risk-based approaches, their foundational frameworks and terminology differ. The FDA regulates Cell and Gene Therapies (CGTs) under a biologics framework, while the EMA governs them as Advanced Therapy Medicinal Products (ATMPs) [1] [11]. A significant divergence lies in the classification of genetically modified cells like CAR-Ts: the EU consistently classifies them as gene therapy products, whereas the US approach can involve more nuanced categorization [1].

The core regulatory principles, however, show convergence. Both agencies emphasize that the "process is the product," meaning that early technical decisions on vector platforms, assay validation, and scale-up methods fundamentally shape regulatory outcomes [4]. Furthermore, both endorse ICH Q9 principles on quality risk management, requiring sponsors to systematically identify, evaluate, and control parameters that impact final product quality [2] [5].

Key Regulatory Differences in Control Strategy Execution

The following table summarizes critical differences in how the FDA and EMA approach specific elements of control strategy, impacting how CPPs and CQAs are defined and managed.

Table 1: Key Regulatory Differences Impacting Control Strategy Development

Regulatory Aspect	FDA (US) Position	EMA (EU) Position
Starting Materials	No formal regulatory definition; uses "critical raw materials" with risk-based control [2].	Precise definition; materials becoming part of the drug substance (e.g., vectors) must follow GMP principles [2] [1].
Viral Vector Classification	Classified as a drug substance, requiring full GMP and facility licensing [2] [11].	Often considered a starting material, especially for ex vivo therapies, though GMP is still expected [2] [11].
Potency Testing for Vectors	Requires a validated functional potency assay for pivotal studies [2].	Infectivity and transgene expression may suffice in early phases; less emphasis on functional assays later [2].
Process Validation (PV) Batches	Number not specified but must be statistically adequate [2].	Generally requires three consecutive batches, with some flexibility [2].
Use of Surrogate Data in PV	Allowed with justification [2].	Allowed only in case of a shortage of starting material [2].
Stability Data for Comparability	Requires thorough assessment, including real-time data for certain changes [2].	Real-time data not always mandatory [2].

Experimental Framework for a Risk-Based Control Strategy

Developing a control strategy requires a systematic, experimental process to define and validate the links between process parameters and product quality. The following workflow provides a methodology acceptable to both major regulators.

Experimental Workflow and Visualization

The foundational experiment for control strategy development is a sequential risk assessment that progresses from initial screening to formal validation. The diagram below outlines this core workflow.

Diagram 1: Experimental Workflow for Control Strategy Development

Phase 1: Defining CQAs and Initial Risk Assessment

Objective: To identify and rank all potential CQAs based on their impact on safety and efficacy, and to link them to relevant manufacturing unit operations.

Protocol:

• Define CQAs from Target Product Profile (TPP): Compile a list of all potential quality attributes from the TPP and quality target product profile (QTPP). Categorize each attribute as Critical (CQA), Key (KQA), or Non-Critical based on the risk of it impacting safety or efficacy. For a CAR-T product, CQAs typically include identity (e.g., CD3+ cell count), potency (e.g., cytotoxic activity), purity (e.g., % of viable cells), and vector copy number [2] [4].
• Perform an Initial Risk Assessment: Use a risk-ranking and filtering tool. Create a matrix with unit operations (e.g., apheresis, activation, transduction, expansion, fill-finish) on one axis and the finalized CQAs on the other. A multi-disciplinary team scores the potential for each unit operation to impact each CQA on a defined scale (e.g., High, Medium, Low). This prioritizes which unit operations require further investigation.

Phase 2: Screening Critical Process Parameters

Objective: To experimentally determine which input process parameters (PPs) within the high-risk unit operations significantly impact the CQAs.

Protocol:

• Design of Experiments (DoE): For a high-risk unit operation like cell transduction, select input PPs (e.g., Multiplicity of Infection (MOI), cell density at transduction, incubation time). Do not use a one-factor-at-a-time approach.
• Execute Screening DoE: A fractional factorial or Plackett-Burman design is suitable for screening. Run the experiments at small scale (e.g., in 24-well plates) that is representative of the process.
• Analyze and Down-select CPPs: Use statistical analysis (e.g., multiple linear regression, Pareto charts) to identify PPs with a significant and meaningful effect on the CQAs. Parameters that do not show a significant effect can be controlled to a set point or range but are not considered critical. Those with a significant effect are designated as Critical Process Parameters (CPPs).

Phase 3: Refining the Linkage and Establishing Controls

Objective: To define the functional relationship between each CPP and its affected CQA(s) and to establish a proven acceptable range (PAR) for each CPP.

Protocol:

• Refined Risk Assessment with FMEA: For the down-selected CPPs, conduct a Failure Mode and Effects Analysis (FMEA). This qualitative analysis scores each CPP based on the Severity of the CQA impact, Occurrence of failure, and Detectability of the failure. The Risk Priority Number (RPN = SxOxD) helps prioritize which CPPs require the most stringent control.
• Characterization DoE: A response surface methodology (e.g., Central Composite Design) is used to model the relationship between the key CPPs and the CQAs. This experiment establishes the functional mathematical relationship and defines the PAR within which CQAs will consistently meet their acceptance criteria.
• Establish Control Methods: For each CPP, define the control strategy, which may include in-process testing (e.g., measuring cell density before transduction), parameter controls on equipment (e.g., temperature range for incubators), and process validation.

Phase 4: Validation and Lifecycle Management

Objective: To validate the control strategy at commercial scale and demonstrate comparability after process changes.

Protocol:

• Process Performance Qualification (PPQ): Execute the locked-down process at commercial scale. The number of batches must be statistically justified for the FDA, while the EMA typically expects three consecutive batches [2]. The data must demonstrate that all CPPs are controlled within their PARs and all CQAs are met.
• Comparability Studies: Following a process change, a comparability exercise is required. The extent of analytical testing (quality attributes), stability studies, and even clinical data increases with the stage of development. Both agencies agree on a risk-based approach for determining the extent of testing needed [2]. The EMA provides specific guidance on attributes to test when changing starting materials, which has no direct FDA equivalent [2].

The Scientist's Toolkit: Essential Reagents and Materials

A successful control strategy relies on high-quality, well-characterized materials. The selection of these materials is itself a critical part of the risk-based approach.

Table 2: Essential Research Reagent Solutions for Control Strategy Development

Reagent/Material	Function in Control Strategy	Critical Considerations & Regulatory Alignment
Cell Starting Materials (e.g., Patient/apheresis material, iPSCs)	The foundational raw material; its quality directly impacts process performance and CQAs.	FDA/EMA Difference: EMA requires some donor testing even for autologous material [2]. Both require careful sourcing and qualification.
Viral Vectors (e.g., Lentivirus, AAV)	Critical for genetic modification; a key source of process and product variability.	FDA/EMA Difference: FDA classifies in-vitro viral vectors as a drug substance; EMA considers them a starting material, impacting GMP requirements [2] [11].
Cell Culture Media & Supplements (e.g., Serum-free media, cytokines, growth factors)	Supports cell growth, viability, and critical functions (e.g., expansion, persistence).	Avoid research-grade materials. Human serum replacement is a key area of development to reduce risk [36]. Quality must increase through clinical phases [1].
Analytical Assays for CQAs (e.g., Flow cytometry, PCR, potency assays)	To measure and monitor CQAs, providing data for risk assessments and DoE.	FDA expects more functional potency assays [2]. Both agencies are open to orthogonal methods (methods using different principles) to build confidence in results [1].
Gene Editing Components (e.g., CRISPR-Cas9 ribonucleoproteins)	For in-situ genetic modification; defines product mechanism of action.	The EMA's updated guideline defines ex vivo genome editing machinery as starting materials, requiring GMP-grade manufacture [1].

Navigating the parallel paths of US and EU regulatory requirements for cell therapy control strategies demands a meticulous, data-driven, and risk-based methodology. While the core scientific principle of linking CPPs to CQAs is universal, successful global developers must account for key differences in starting material definitions, viral vector classification, and potency testing expectations. The experimental framework outlined here—progressing from risk assessment and DoE to validation and comparability—provides a robust structure for building a control strategy that satisfies both the FDA and EMA. By embedding these practices early in development and leveraging a well-characterized toolkit of reagents and assays, researchers can mitigate the significant CMC-related risks that currently stall many promising therapies, thereby accelerating the delivery of transformative treatments to patients worldwide.

For developers of cell and gene therapies (CGTs), manufacturing changes are inevitable throughout the product lifecycle, driven by process optimization, scale-up, or supply chain adaptations. Comparability exercises serve as the critical scientific bridge that connects pre- and post-change products, demonstrating that modifications do not adversely impact safety, purity, or efficacy. Within the broader thesis of comparing EU and US cell therapy Chemistry, Manufacturing, and Controls (CMC) requirements, managing these changes presents a complex regulatory challenge. The European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) share the fundamental principle that the "process is the product," especially for complex biologics where manufacturing changes can alter critical quality attributes [4]. However, regulatory nuances between regions demand carefully tailored strategies. A striking analysis of FDA's Complete Response Letters (CRLs) from 2020 to 2024 revealed that 74% cited manufacturing or quality (CMC) deficiencies, underscoring the pivotal importance of robust comparability planning for successful global market entry [4].

Regulatory Framework: Comparing US and EU Requirements

Foundational Principles and Guidelines

Currently, CGTs are generally considered outside the direct scope of the ICH Q5E guideline on comparability, though a new annex specific to these products is in development [2]. In the interim, sponsors must navigate regional guidances that, while aligned on core principles, exhibit key differences in emphasis and application.

• EU Perspective: The EMA provides a structured framework through its 'Questions and Answers on comparability considerations for advanced therapy medicinal products (ATMP)' and multidisciplinary guidelines for genetically modified cells. A specific guideline for demonstrating comparability for CGTs in clinical development becomes effective in July 2025 [2]. The EMA emphasizes that for medicinal products containing genetically modified cells, specific attributes must be evaluated when changing the manufacturing process for recombinant starting materials. These include full vector sequencing, confirming the absence of replication competent virus (RCV), comparing impurities, and assessing stability [2].

• US Perspective: The FDA has issued draft guidance on comparability for CGT products (July 2023), which reflects the agency's current thinking. While comprehensive, some observers note that future drafts could benefit from more explicitly addressing comparability requirements following changes related to recombinant starting materials [2].

Key Areas of Regulatory Nuance

The following table summarizes critical differences in regulatory approaches to comparability between the EU and US that CGT developers must incorporate into their strategic planning.

Table 1: Key Regulatory Differences in EU and US Comparability Requirements

Regulatory Aspect	FDA (US) Position	EMA (EU) Position
Stability Data for Comparability	Prefers thorough assessment including real-time data for certain changes [2]	Real-time data not always mandatory; may accept other evidence [2]
Use of Historical Data	Inclusion of historical data is recommended to support comparability conclusions [2]	Comparison to historical data is neither required nor routinely recommended [2]
Potency Testing for Viral Vectors	Expects more functional assays; validated functional potency assay is essential for pivotal studies [2]	Infectivity and transgene expression often sufficient in early phase; different expectations for functional assays [2]
RCV Testing for GM Cells	Requires testing of the cell-based drug product itself, in addition to the vector [2]	If absence of RCV is demonstrated on the vector, further testing on the final genetically modified cells is not required [2]
Process Validation Batches	Number not specified but must be statistically adequate based on process variability [2]	Generally expects three consecutive batches, though allows some flexibility [2]

Designing a Phase-Appropriate Comparability Study

A one-size-fits-all approach to comparability is ineffective. The depth of investigation should be phase-appropriate, scaling in rigor as the product advances toward marketing authorization. The overall intention is to provide regulators with a transparent pathway from the safety, efficacy, and quality data from pre-change clinical batches to post-change batches [37].

Analytical Toolkit for Comparability Exercises

A robust comparability package relies on a suite of analytical studies that go beyond routine release testing. These studies are designed to reveal subtle differences in product attributes that might not be detected by standard methods.

Table 2: Core Components of a Comparability Analytical Package

Study Type	Purpose	Key Methodologies
Extended Characterization	Provides an orthogonal, deeper understanding of the molecule's intrinsic properties, especially Critical Quality Attributes (CQAs) [37]	- Peptide mapping (LC-MS)- Size Variant Analysis (SEC-MALS)- Charge Variant Analysis (iCIEF, CEX)- Glycan analysis- Sequence Variant Analysis (SVA)
Forced Degradation Studies	"Pressure-tests" the molecule to uncover and compare degradation pathways under stress conditions, informing product stability and shelf-life [37]	- Thermal stress (e.g., 25°C, 40°C)- pH stress (e.g., low and high pH)- Oxidative stress (e.g., hydrogen peroxide)- Light stress (per ICH guidelines)
Stability Studies	Demonstrates that the post-change product maintains quality attributes over time under recommended storage conditions [37]	- Real-time stability under recommended storage conditions- Accelerated stability studies

The following workflow outlines the strategic progression of a comprehensive comparability exercise, from planning to regulatory submission.

Diagram 1: Strategic Workflow for Comparability Exercises

Phase-Appropriate Implementation

The scale and rigor of comparability testing should evolve with the product's stage of development.

• Early Phase (e.g., Phase 1 IND): At this stage, knowledge is still accumulating. It is often acceptable to use single batches of pre- and post-change material for head-to-head biophysical characterization using platform methods [37]. Forced degradation studies can be used as a screening tool to understand the molecule's behavior and inform analytical method development.

• Late Phase (e.g., Phase 3 to BLA/MAA): The evidence standard is significantly higher. A typical package should include head-to-head testing of multiple pre- and post-change batches (e.g., 3 vs. 3) [37]. The analytical methods should be more molecule-specific, fully validated, and focused on established CQAs. The goal is to provide definitive evidence that the change does not impact the safety or efficacy profile of the product.

The Scientist's Toolkit: Essential Reagents and Materials

Successful execution of a comparability study depends on high-quality, well-characterized materials and reagents. Proper planning of their procurement and qualification is fundamental.

Table 3: Essential Research Reagents for Comparability Studies

Reagent/Material	Function in Comparability	Critical Considerations
Reference Standard (RS)	Serves as the benchmark for quality attributes for both pre- and post-change material [37]	- Must be well-characterized and stable- Qualification and stability should be documented
Pre-Change Drug Substance Lots	Acts as the baseline for comparison [37]	- Select recent, representative batches that passed release- Avoid "cherry-picking" to ensure data integrity
Post-Change Drug Substance Lots	The test material for the comparability exercise [37]	- Should be manufactured as close as possible to pre-change batches to avoid age-related differences
Characterized Cell Banks	Ensures consistent starting materials for manufacturing both pre- and post-change batches [17]	- Requires thorough qualification and testing for adventitious agents
Critical Raw Materials	Materials (e.g., cytokines, growth factors, vectors) whose variation could impact product quality [2] [17]	- Implement enhanced control strategies aligned with risk- Quality and sourcing must be consistent or justified

Strategic Implementation and Global Submissions

Navigating the regulatory landscape for comparability requires more than just technical excellence; it demands forward-thinking strategy and meticulous documentation.

Proactive Planning and Risk Management

• Engage Regulators Early: Both the FDA and EMA encourage early dialogue. The FDA's Expedited Programs draft guidance "strongly" encourages sponsors to discuss CMC readiness, including manufacturing challenges, through available interaction pathways [38].
• Develop a Comparability Protocol Prospectively: A前瞻性 comparability protocol outlines the studies and acceptance criteria before implementing a change. The FDA has guidance on this topic, and having an agreed-upon protocol can streamline the review of post-approval changes [17] [39].
• Leverage Platform Data: Where similar manufacturing steps are used across products, both FDA and EMA accept the use of platform data to support process validation and, by extension, certain comparability assessments [2].

Documentation and Submission Strategy

The final comparability package must tell a compelling scientific story. The report should transparently present the rationale for the change, the study design, and a detailed interpretation of the data, explaining any observed differences [37]. For global programs, it is crucial to tailor the submission to address region-specific requirements highlighted in Table 1. For instance, a submission to the EMA for a product containing genetically modified cells must include specific tests on the finished product, such as transduction efficiency, vector copy number, and transgene expression, for which there is no direct FDA equivalent [2].

Successfully managing manufacturing changes through robust comparability exercises is a cornerstone of global CGT development. While the US and EU regulatory systems are fundamentally aligned on the need for rigorous science and a risk-based approach, strategic success hinges on a deep understanding of their nuanced differences in areas like stability data, potency testing, and historical data usage. By adopting a phase-appropriate strategy, employing a comprehensive analytical toolkit, and proactively engaging with both agencies, sponsors can construct defensible comparability packages. This disciplined approach not only facilitates regulatory approval across jurisdictions but also strengthens the overall control strategy, ensuring that life-changing therapies can reach patients consistently and without compromise.

Potency assays are fundamental quantitative measures of the biological activity of Cell Therapy Products (CTPs) and Advanced Therapy Medicinal Products (ATMPs), serving as a critical link between product quality and clinical efficacy [40]. They are required by regulation to ensure that each product batch possesses the specific ability or capacity to effect its intended therapeutic result [41] [40]. For cell and gene therapies, a successful potency assay must be mechanistically linked to the product's biological effect, precise, and accurate, reflecting the complex functional attributes of living cellular drugs [41].

The development of these assays presents unique challenges due to the inherent variability of biological systems, complex mechanisms of action (MoA), and stringent regulatory requirements across different regions [41]. This guide objectively compares the experimental methods and regulatory expectations for functional potency assays between the United States (US) and European Union (EU), providing researchers with structured data and protocols to navigate the global development landscape.

Regulatory Landscape: A Comparative Framework of US and EU Requirements

While both US and European regulators mandate potency testing, key differences in approach and emphasis exist, influencing assay development strategy.

Table 1: Key Regulatory Differences in Potency Testing Requirements between the US and EU

Regulatory Aspect	US FDA Position	EU EMA Position
Primary Potency Assay	Requires a quantitative functional potency assay for lot release [41] [40].	May allow validated surrogate assays for release if a functional characterization assay exists and correlation is demonstrated [40].
Assay Validation	Full validation required for commercial release, following ICH Q2(R2) [41].	Alignment with ICH Q2(R2), with a phase-appropriate approach for clinical trials [41].
Potency for Viral Vectors (in vitro use)	Expects more functional assays; classified as a drug substance [2].	Infectivity and transgene expression may be sufficient, especially in early phases; often considered a starting material [2].
Mechanism of Action (MoA)	Assay must reflect the product's specific MoA; a combination of assays may be needed for complex MoAs [41] [42].	Similarly emphasizes the importance of MoA-reflective assays [40].
Starting Materials	No formal regulatory definition; employs an enhanced risk-based control approach [2] [1].	Precisely defines 'starting materials' (e.g., vectors, cells) with GMP principles often applied earlier [2] [1].

Beyond these specific testing differences, the fundamental regulatory categorization varies. In the US, these products are regulated as Cell and Gene Therapies (CGTs) under the Center for Biologics Evaluation and Research (CBER), while in the EU, they are classified as Advanced Therapy Medicinal Products (ATMPs) [11] [1]. This influences the governing guidelines and, in some cases, the level of oversight for certain components like viral vectors [2] [11].

Quantitative Analysis of Approved Therapy Potency Tests

An analysis of potency tests for 31 US FDA-approved CTPs reveals the practical application of these regulatory principles. This data provides a benchmark for developers designing their own assay strategies.

Table 2: Analysis of Potency Tests Used in 31 US FDA-Approved Cell Therapy Products (2010-2024) [43]

Category of Potency Test	Number of Tests (Non-Redacted)	Percentage of Total	Examples of Specific Methods
Viability and Count	37	52%	Total Nucleated Cells (TNC), Viable CD34+ cell count, Cell viability [43]
Expression	19	27%	CAR expression by flow cytometry, Vector Copy Number (qPCR) [43]
Bioassays	7	7%	IFN-γ release upon target cell stimulation, Cytotoxic activity [43]
Genetic Modification	6	9%	Colony Forming Units (CFU), Percent LVV+ cells [43]
Histology	2	3%	Tissue organization, cell viability in tissue context [43]

This analysis shows that while direct functional bioassays are used in only 23% of products with non-redacted data, over 84% of CTPs cite physicochemical or other non-bioassay methods as part of their potency testing, often employing a combination of methods [43]. For example, the CAR-T therapy Kymriah utilizes both CAR expression (Expression) and IFN-γ release in response to CD19+ cells (Bioassay) as potency tests [43]. This underscores the regulatory expectation for MoA-reflective assays, even when surrogates are used for routine release.

Experimental Protocols for Functional Potency Assays

Below are detailed methodologies for key functional potency assays commonly used for cell-based therapies, particularly those with cytotoxic MoAs like CAR-T and TCR-T cells.

HiBiT Target Cell Killing Bioassay (Gain of Signal)

This platform measures the specific lytic activity of effector cells (e.g., CAR-T) against target cells.

Detailed Protocol:

• Cell Preparation:
  • Engineer target cells (e.g., tumor cells) to stably express a cell membrane-bound HiBiT fusion protein [42].
  • Expand and harvest effector cells (e.g., CAR-T cells).
• Co-culture Setup:
  • Seed target cells in a tissue culture-treated microplate.
  • Add serially diluted effector cells to the target cells at various Effector to Target (E:T) ratios. Include target cell-only controls (for maximum signal) and effector cell-only controls (for background) [42].
  • Centrifuge the plate briefly to initiate cell contact and incubate for 4-72 hours at 37°C, 5% CO₂, depending on the kinetics of killing.
• Signal Detection:
  • Following incubation, add a prepared reagent containing the complementary LgBiT protein and the luciferase substrate to the culture well [42].
  • Upon target cell lysis by the effector cells, the HiBiT fusion protein is released into the supernatant. It binds to LgBiT in the reagent, forming a functional NanoLuc Luciferase enzyme.
  • Measure the resulting bright, luminescent signal using a luminometer. The signal intensity is directly proportional to the number of target cells lysed [42].
• Data Analysis:
  • Calculate % Cytotoxicity using the formula: (Experimental Signal – Target Cell Control Signal) / (Target Cell Control Signal) * 100 [42].
  • Plot % Cytotoxicity against E:T ratio or effector cell concentration to generate a dose-response curve and calculate relative potency (e.g., EC₅₀).

Luciferase-Based Cytotoxicity Assay (Loss of Signal)

A complementary method measuring the decrease in signal as engineered target cells are killed.

Detailed Protocol:

• Cell Engineering:
  • Stably transduce target cells to express a luciferase reporter gene, such as Firefly luciferase [42].
• Co-culture and Assay:
  • Seed luciferase-expressing target cells in a microplate.
  • Add effector cells at varying E:T ratios, similar to the HiBiT protocol.
  • After the co-culture incubation period, add a homogeneous luciferase assay reagent (e.g., Bright-Glo or One-Glo) directly to the wells [42].
  • Lyse the cells according to the reagent's protocol and incubate to allow the luciferase reaction to reach steady state.
• Measurement and Analysis:
  • Measure the luminescent signal. A decrease in signal relative to the target cell-only control indicates cytotoxicity.
  • Calculate % Cytotoxicity: 100 - [(Experimental Signal / Target Cell Control Signal) * 100].

T Cell Activation Bioassay (NFAT Reporter Assay)

This assay is used to screen and validate the function of new TCRs or CARs by measuring proximal T-cell signaling events.

Detailed Protocol:

• Effector Cell Engineering:
  • Utilize engineered T cell lines (e.g., TCR/CD3 Effector Cells) that contain an NFAT-responsive element driving the expression of a luciferase reporter gene [42].
• Transduction/Transfection:
  • Introduce the candidate CAR or TCR construct into the reporter effector cells via lentiviral transduction or transfection.
• Antigen-Specific Activation:
  • Co-culture the transduced effector cells with antigen-presenting target cells. For specificity validation, use isogenic target cells with and without the target antigen (e.g., CD19-knockout) [42].
  • Incubate for ~6 hours to allow for CAR:target engagement and subsequent intracellular signaling.
• Signal Detection:
  • Add a luciferase assay reagent to the culture.
  • Measure luminescence. The signal intensity is directly proportional to the degree of TCR or CAR activation upon antigen recognition [42].
• Specificity and Stability Assessment:
  • Specificity: Demonstrate that luminescence is only induced in the presence of the correct antigen [42].
  • Stability-Indicating: Use the assay to test forced degradation samples (e.g., heat-treated lentivirus), which should show a loss of potency (increased EC₅₀ or decreased maximum response) [42].

The Scientist's Toolkit: Essential Research Reagent Solutions

Selecting the right tools is critical for developing robust and reproducible potency assays. The following table details key reagents and their functions.

Table 3: Key Research Reagents for Potency Assay Development

Reagent / Solution	Function in Potency Assays	Example Application
Lumit Cytokine Immunoassays	Homogeneous, "no-wash" luminescent immunoassays for cytokine detection (e.g., IFN-γ) directly in cell culture [42].	Quantifying CAR-T cell activation by measuring IFN-γ secretion upon co-culture with target cells [42].
HiBiT Target Cell Killing Bioassay System	A gain-of-signal platform for real-time, specific measurement of cell-mediated cytotoxicity [42].	Quantifying potency of CAR-T, TCR-T, or CAR-NK cells based on their ability to lyse HiBiT-tagged target cells [42].
Bright-Glo / One-Glo Luciferase Assay Systems	Homogeneous, robust reagents for detecting Firefly luciferase activity, used in loss-of-signal cytotoxicity assays [42].	Measuring cytotoxicity against target cells engineered to stably express Firefly luciferase [42].
T Cell Activation Bioassay (NFAT Reporter)	Engineered T-cell line with an NFAT-luciferase reporter for measuring TCR or CAR activation [42].	Screening and validating the function of novel CAR or TCR constructs in a high-throughput format [42].
SoftMax Pro Software	Data analysis software with advanced statistical tools for processing bioassay data, including outlier analysis and relative potency calculations, supporting FDA 21 CFR Part 11 compliance [41].	Determining relative potency using 4-parameter logistic (4PL) curve fit, assessing parallelism, and generating GxP-compliant reports [41].

Developing functional, mechanistically linked potency assays is a cornerstone of successful cell therapy development. The experimental data and protocols presented here demonstrate that a thorough understanding of the product's MoA is paramount in selecting the appropriate bioassay format, whether it be a target cell killing assay, a T-cell activation reporter assay, or cytokine secretion measurement.

Navigating the nuanced differences between US and EU regulatory expectations requires a strategic, phase-appropriate approach. While the FDA mandates a quantitative functional assay for release, the EMA may offer some flexibility with surrogate assays, provided a functional link is firmly established. By leveraging modern, sensitive tools like the HiBiT and reporter assays, and by building a comprehensive potency profile early in development, researchers can create robust control strategies that meet global regulatory standards, ensuring the consistent quality, safety, and efficacy of these transformative therapies for patients worldwide.

In the development of cell and gene therapies (CGTs), known as advanced therapy medicinal products (ATMPs) in the European Union, the initial stages of donor eligibility assessment and cell collection form the foundational pillar of the entire supply chain. These starting materials substantially influence the quality, safety, and efficacy of the final therapeutic product, making regulatory-compliant handling imperative for global development programs [2] [1]. The complex and fragmented nature of the cell and gene therapy supply chain, valued at $1.5 billion in 2024 and predicted to reach $4.4 billion by 2034, necessitates specialized logistics solutions supporting patient identification, cell collection, therapeutic processing, and long-term outcome monitoring [44]. For allogeneic or "off-the-shelf" therapies particularly, which rely on large, well-characterized donor pools to treat multiple patients, disruptions in donor sourcing directly threaten manufacturing timelines and patient access to life-saving treatments [45]. This guide objectively compares the regulatory requirements and operational approaches for donor eligibility and cell collection between the United States (US) and European Union (EU), providing researchers and drug development professionals with actionable insights for global CMC planning.

Regulatory Framework Comparison: US vs. EU

Governing Structures and Key Legislation

The regulatory frameworks governing donor eligibility and cell collection differ substantially between the US and EU, reflecting distinct historical approaches to biologics regulation.

United States Framework: In the US, cell and gene therapies are regulated primarily as biologics by the Food and Drug Administration (FDA) under Title 21 of the Code of Federal Regulations (CFR) [19] [11]. Human cells, tissues, and cellular and tissue-based products (HCT/Ps) specifically fall under 21 CFR Part 1271, which establishes eligibility requirements for donors through subpart C [2]. The FDA's Center for Biologics Evaluation and Research (CBER) oversees these products through its Office of Therapeutic Products (OTP), which provides comprehensive guidance for industry covering donor eligibility determination, screening, testing, and cell collection procedures [19] [1].

European Union Framework: In the EU, ATMPs fall under Regulation (EC) No 1394/2007, with donor eligibility and cell collection requirements primarily governed by the EU Tissue and Cells Directives (EUTCD) and the newly revised legislation for substances of human origin (SoHOs) [2] [1]. The European Medicines Agency (EMA) Committee for Advanced Therapies (CAT) provides scientific recommendations for classification and oversight, with national competent authorities implementing requirements [5] [1]. The updated SoHO legislation, revised in 2024, brings blood products, tissues, and cells under a single regulation to provide greater protection to patients and donors, extending activities covered to include donor registration, collection, testing, storage, distribution, import, and export [1]. All parties handling these activities must comply with the new regulation by 7 August 2027 [1].

Table 1: Key Regulatory Framework Differences

Regulatory Aspect	United States (US)	European Union (EU)
Primary Legislation	21 CFR Part 1271 (HCT/Ps) [19]	SoHO Legislation (2024) [1]
Governing Body	FDA CBER/OTP [19] [1]	EMA CAT & National Authorities [5] [1]
Donor Testing Labs	CLIA-accredited laboratories [2]	Licensed premises & accredited centres [2]
GMP Application	Phase-appropriate approach [1]	GMP required for investigational products in first-in-human studies [1]
Importation Requirements	FDA compliance [1]	EU GMP certification required, site inspection or mutual recognition [1]

Donor Eligibility and Testing Requirements

Both US and EU regulatory frameworks mandate comprehensive donor screening and testing to prevent transmission of infectious diseases, though specific requirements and implementation differ.

US Donor Testing Requirements: FDA standards require comprehensive testing panels under 21 CFR 1271 for HIV-1 and HIV-2, Hepatitis B (HBV), Hepatitis C (HCV), Human T-lymphotropic virus (HTLV) types I and II, syphilis, Cytomegalovirus (CMV), and West Nile Virus (WNV) [45]. These tests must be performed in CLIA-accredited laboratories, ensuring standardized testing quality and reliability across collection facilities [2]. For autologous donations, where cells are collected from and administered to the same patient, the FDA maintains specific requirements to ensure product safety and quality [2].

EU Donor Testing Requirements: The EMA governs donor testing through the EUTCD, which requires testing to be handled in licensed premises and accredited centers [2]. The EU requires some donor testing even for autologous material, extending safety standards beyond the US approach [2]. The newly implemented SoHO legislation further harmonizes these requirements across member states while maintaining specific national add-ons in some cases [1]. The EU system utilizes a Qualified Person (QP) who must certify that appropriate quality standards have been met before product release [2].

Operational Advantage of US Donor Ecosystem: The US donor pool offers significant operational advantages for global development, featuring genetic diversity that enables matches for every ancestry and specialized HLA needs, a robust regulatory environment supporting safe and ethical sourcing, and operational scalability facilitating rapid donor recruitment and high-volume manufacturing partnerships [45]. US laboratories routinely validate combined FDA, EMA, and other international panels (such as Japan's PMDA), enabling single US collections to meet global requirements and speeding delivery while protecting patients from delays [45].

Table 2: Comparative Donor Testing Requirements

Testing Parameter	United States (US)	European Union (EU)
HIV-1/HIV-2	Required [45]	Required
Hepatitis B (HBV)	Required [45]	Required
Hepatitis C (HCV)	Required [45]	Required
HTLV I/II	Required [45]	Required (where applicable)
Treponema pallidum (Syphilis)	Required [45]	Required
Cytomegalovirus (CMV)	Required [45]	Required (where applicable)
West Nile Virus (WNV)	Required [45]	National requirements may apply
Autologous Donor Testing	Specific requirements [2]	Required in some cases [2]

Experimental Protocols and Methodologies

Donor Eligibility Determination Protocol

Objective: To establish standardized procedures for determining donor eligibility based on US and EU regulatory requirements, ensuring consistent application across collection sites.

Materials and Reagents:

• Donor medical history questionnaire (approved by Institutional Review Board)
• Physical assessment equipment
• Blood collection kits (containers, needles, tourniquet, antiseptics)
• Sample tubes (serum, plasma, whole blood)
• Infectious disease testing kits (FDA-approved/CE-marked)
• Chain of identity documentation forms
• Temperature-monitored shipping containers

Methodology:

• Donor Identification and Consent: Obtain informed consent using institution-approved documentation explaining the donation process, tests performed, and potential health implications.
• Medical History Interview: Conduct comprehensive interview assessing:
  • History of transmissible diseases
  • Risk factors for HIV, HBV, HCV, HTLV, and other relevant pathogens
  • Travel history to regions with endemic infections (e.g., malaria, Zika)
  • Medication history and exposure to biologics
  • Family history of neurodegenerative disorders
• Physical Assessment: Perform examination to identify physical signs of:
  • Relevant transmissible diseases
  • Conditions that might contraindicate donation
  • General health status evaluation
• Blood Collection: Draw samples for required infectious disease testing using aseptic technique:
  • Collect appropriate volume based on testing requirements
  • Utilize proper sample handling procedures to maintain integrity
  • Label samples with unique identifier matching donor records
• Infectious Disease Testing: Perform testing using FDA-approved/CE-marked test kits:
  • HIV-1/HIV-2: Nucleic acid testing (NAT) and serological tests
  • HBV: HBsAg, anti-HBc, HBV NAT
  • HCV: Antibody testing and NAT
  • Other required testing based on regional requirements
• Donor Eligibility Determination: Review all collected information against regulatory criteria:
  • Document eligibility decision with justification
  • Implement process for managing deferred donors
  • Maintain complete records per regulatory requirements

Quality Control: Implement external proficiency testing programs, internal quality control samples with each assay run, equipment calibration and maintenance logs, and personnel training and competency assessment documentation.

Cell Collection and Initial Processing Protocol

Objective: To standardize cell collection procedures (leukapheresis, bone marrow aspiration, tissue donation) for cell therapy manufacturing, maintaining cell viability, potency, and microbial control.

Materials and Reagents:

• Leukapheresis system or bone marrow aspiration kits
• Anticoagulants (heparin, ACD-A)
• Cell processing reagents (buffers, media, enzymes)
• Cryopreservation solutions (DMSO, serum/media)
• Sterile collection containers and transfer sets
• Temperature monitoring and control equipment
• Microbial contamination testing kits

Methodology:

• Collection Site Preparation:
  • Verify facility meets environmental monitoring requirements
  • Calibrate and qualify equipment
  • Prepare aseptic processing area
• Cell Collection:
  • Leukapheresis: Perform using standardized protocols targeting specific cell populations
  • Bone Marrow Aspiration: Utilize sterile surgical technique with appropriate anesthesia
  • Tissue Collection: Follow aseptic technique with minimal manipulation
• Anticoagulation: Use approved anticoagulants at validated ratios to maintain cell viability while preventing clotting.
• Initial Processing:
  • Perform cell count and viability assessment
  • Conduct volume reduction if necessary
  • Implement initial separation techniques if required
• Preservation and Shipping:
  • Cryopreserve using controlled-rate freezing if not processing immediately
  • Package according to chain of custody requirements
  • Implement temperature monitoring during transport
• Microbial Control:
  • Perform sterility testing per pharmacopoeial standards
  • Conduct endotoxin testing
  • Implement Gram stain or rapid microbial methods when appropriate

Data Analysis: Document total nucleated cell count, cell viability percentage, cell composition (CD34+ count for hematopoietic cells), collection volume, sterility results, and shipment conditions (time, temperature, integrity upon receipt).

Workflow Visualization: Global Donor Management

The following diagram illustrates the integrated workflow for managing donor eligibility and cell collection across US and EU regulatory frameworks:

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 3: Essential Reagents and Materials for Donor Eligibility and Cell Collection Research

Tool/Reagent	Function/Application	Regulatory Considerations
Nucleic Acid Testing (NAT) Kits	Detection of viral pathogens (HIV, HBV, HCV, WNV) with high sensitivity during donor screening [45]	Must be FDA-approved or CE-marked; validated per regulatory standards
Serological Testing Assays	Detection of pathogen-specific antibodies for comprehensive infectious disease screening [45]	Required for complete donor eligibility determination in both US and EU
Cell Separation Media	Density gradient separation of mononuclear cells from whole blood or apheresis products	Must be GMP-grade for clinical use; research grade may be acceptable for early development
Cryopreservation Solutions	Preservation of cell viability during frozen storage and transport	DMSO concentration and serum content must be optimized and validated for specific cell types
Sterility Testing Kits	Microbial contamination assessment per pharmacopoeial standards (USP, Ph. Eur.)	Required for all cellular starting materials; must use validated methods
Cell Viability Assays	Determination of cell viability and potency post-collection and during processing	Functional assays expected by FDA; phase-appropriate validation required
Chain of Identity Systems	Maintenance of donor-recipient linkage through barcoding or electronic systems	Critical for patient safety; must maintain integrity throughout supply chain

Navigating donor eligibility and cell collection requirements across the US and EU presents both challenges and opportunities for cell therapy developers. The US donor ecosystem offers advantages in genetic diversity, regulatory reliability, and operational scalability, with testing infrastructure capable of meeting global requirements through single collections [45]. Meanwhile, the EU's evolving regulatory landscape, particularly the new SoHO legislation, aims to harmonize standards while maintaining rigorous safety protocols [1]. For developers pursuing global approval pathways, early engagement with regulators through INTERACT meetings with FDA CBER or classification requests with the EMA CAT is essential to clarify region-specific requirements [1]. Understanding these nuanced differences in donor eligibility assessment, testing methodologies, and collection standards enables more efficient global development strategies, ultimately accelerating patient access to transformative cell and gene therapies worldwide.

Overcoming Common Hurdles: CMC Pitfalls and Proactive Solutions for Cell Therapies

For developers of cell and gene therapies, potency testing represents one of the most significant Chemistry, Manufacturing, and Controls (CMC) challenges. A potency assay is not merely a quality control test; it is a direct measure of a therapy's biological activity and should correlate with its intended clinical effect [46]. Regulators require developers to measure the potency of all biologics, including gene and cell therapies to ensure that a consistent product is delivered to all patients [47]. The fundamental challenge lies in designing assays that accurately reflect the complex mechanism of action (MoA) of these advanced therapies, while also meeting regulatory expectations across different jurisdictions.

The complexity of this task is magnified for global development programs, where the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) demonstrate nuanced differences in their regulatory approaches. While both agencies agree that potency assays must be linked to the product's MoA, their specific expectations regarding assay validation, timing of implementation, and acceptable methodologies can diverge [2] [1]. Understanding these distinctions is crucial for researchers and drug development professionals navigating the intricate pathway from preclinical development to commercial approval.

Regulatory Landscape: US vs EU Perspectives

Comparative Analysis of Key Regulatory Requirements

The regulatory frameworks governing potency testing for cell therapies, known as Cell and Gene Therapies (CGTs) in the US and Advanced Therapy Medicinal Products (ATMPs) in the EU, show both convergence and divergence in critical areas [2] [11].

Table: Comparison of FDA and EMA Regulatory Requirements for Potency Testing

Regulatory Aspect	FDA Position	EMA Position
Potency Testing for Viral Vectors for In Vitro Use	Validated functional potency assay essential to assess efficacy of drug product used in pivotal studies [2]	Infectivity and transgene expression generally sufficient in early phase; less functional assays acceptable later [2]
Timing of Potency Assay Implementation	Recommended to be included among release tests starting in Phase I studies [18]	Phase-appropriate approach with increasing rigor through development [1]
Assay Validation Requirements	Full validation required under ICH Q2(R2) by Phase 3 and pre-registration [1]	Validated methods encouraged but not strictly required for early phases; orthogonal testing bolsters confidence [1]
Use of Alternative Methodologies	Openness to accepting alternative methods where feasible and justifiable; case-by-case acceptance of New Approach Methodologies (NAMs) [1]	Explicit statement that orthogonal methods should be considered for analytical testing to ensure robustness [1]
Potency Assay Approach	Expectation of functional, biologically relevant assays that reflect the mechanism of action [1] [46]	Recognition that multiple biological activities may require a potency assay matrix for complex products [18]

Analysis of Regulatory Trends

The regulatory environment for potency testing is characterized by increasing rigor from both agencies, with the FDA particularly emphasizing the need for potency assays to be implemented early in clinical development [18] [46]. A significant challenge cited by developers is that the MoA may not be fully understood at early development stages, making it difficult to establish a definitive potency assay [18]. Both regulators now recognize that for therapies with multimodal mechanisms, a single potency assay may be insufficient, necessitating a matrix of assays that collectively reflect the product's biological activity [18].

The FDA has identified potency testing as one of the most common CMC deficiencies in CGT programs, frequently issuing Complete Response Letters (CRLs) for inadequate potency methods [46]. The agency expects potency assays to be quantitative, stability-indicating, and demonstrate lot-to-lot consistency [48]. Meanwhile, the EMA's recently updated guideline on quality requirements for ATMPs emphasizes that orthogonal methods (using different scientific principles to measure the same attribute) should be considered to ensure robustness, particularly when reference standards or validated methods are lacking [1].

Technical Challenges in MoA-Linked Potency Assay Development

Mechanism of Action Definition

The foundation of any successful potency assay is a clearly defined MoA. For cell therapies, this often involves multiple biological processes that must be captured in the potency assessment. A CAR-T cell therapy, for instance, typically requires assays measuring:

• Target cell binding through specific antigen recognition
• Immune synapse formation and T-cell activation
• Cytokine production and secretion
• Direct cytolytic activity against target cells [33]

The complexity increases for engineered macrophage therapies or other multimodal products, where the MoA may involve several distinct biological activities that must be reflected in a comprehensive potency matrix [18].

Analytical Development Considerations

Translating MoA understanding into robust analytical methods presents several technical challenges:

• Functional vs. Identity Assays: Many developers mistakenly rely on identity markers (e.g., vector copy number, transduction efficiency) as surrogate potency measures, rather than developing true functional assays that reflect biological activity [2] [46].
• Matrix Approach: For complex therapies with multimodal mechanisms, a single assay is often insufficient. Regulators increasingly expect a potency assay matrix that collectively captures the key biological functions [18].
• Quantitative Design: The ideal potency assay should be quantitative rather than qualitative, allowing for precise measurement of biological activity and establishing meaningful specifications [48].
• Stability-Indicating Capability: Potency assays must be able to detect product degradation over time, requiring careful design to measure the most labile aspects of biological activity [46].

Diagram 1: Multi-Attribute Potency Assay Development Workflow. This diagram illustrates the comprehensive approach required for developing potency assays that capture complex mechanisms of action, particularly for multimodal cell therapies.

Experimental Approaches and Methodologies

Establishing a Comprehensive Potency Assurance Strategy

Developing a robust potency strategy requires systematic approach that begins early in product development. The following experimental framework provides a methodology for establishing MoA-linked potency assays:

Phase 1: MoA Deconstruction and Critical Quality Attribute Identification

• Comprehensive Literature Review: Analyze existing knowledge about similar therapeutic modalities and their biological effects
• Pathway Mapping: Identify key signaling pathways and biological processes implicated in the therapy's intended function
• In Vitro Functional Screens: Conduct preliminary assays to correlate specific cellular activities with desired therapeutic outcomes
• CQA Prioritization: Identify critical quality attributes that most directly influence the product's biological activity [49] [46]

Phase 2: Assay Design and Development

• Orthogonal Method Selection: Choose multiple assay formats that measure the same attributes using different scientific principles (e.g., flow cytometry, ELISA, functional co-culture)
• Reference Standard Establishment: Create well-characterized reference materials for assay calibration and comparison
• Matrix Configuration: Determine the appropriate combination of assays that collectively reflect the complete MoA
• Precision and Accuracy Assessment: Conduct preliminary testing to establish assay performance characteristics [1] [48]

Phase 3: Assay Qualification and Validation

• Specificity Testing: Demonstrate the assay measures the intended attribute without interference
• Linearity and Range Determination: Establish the quantitative relationship between signal and analyte concentration
• Robustness Evaluation: Assess assay performance under varying conditions (different operators, equipment, reagents)
• Stability-Indicating Verification: Confirm the assay can detect product degradation over time [1] [46]

Research Reagent Solutions for Potency Assay Development

Table: Essential Research Reagents for Cell Therapy Potency Assay Development

Reagent/Category	Function in Potency Testing	Specific Applications
GMP-Grade Cytokines & Growth Factors	Provide standardized stimulation for functional assays	T-cell activation, differentiation assessment [46]
Quality-Controlled Cell Lines	Serve as target cells in cytotoxicity assays	Standardized potency measurement across batches [49]
Flow Cytometry Antibody Panels	Enable quantification of cell surface and intracellular markers	Phenotype characterization, activation status [18]
Multiplex Cytokine Detection Kits	Simultaneous measurement of multiple secreted factors	Comprehensive immune response profiling [18]
Reference Standard Materials	Calibrate assays and enable cross-batch comparison	Maintain consistency throughout development [48]
Cell Culture Media & Supplements	Support maintenance of sensitive primary cells	Ensure consistent assay performance [49]

Case Studies and Experimental Data

CAR-T Cell Therapy Potency Assay Implementation

Background: A sponsor developing a CD19-targeting CAR-T therapy established a comprehensive potency matrix to address the complex MoA involving T-cell activation, proliferation, and cytotoxicity.

Experimental Approach: The team implemented three complementary assays:

• Flow Cytometry-Based Binding Assay: Quantified CD19 binding specificity using recombinant antigen
• Cytokine Release Assay: Measured IFN-γ and IL-2 production upon antigen exposure
• Real-Time Cytotoxicity Assay: Monitored target cell killing using impedance-based technology

Results and Regulatory Outcome: The initial BLA submission included only the binding and cytokine assays. The FDA issued a CRL noting the absence of a functional cytotoxicity assay, which was considered essential for reflecting the complete MoA. After implementing the triple-assay matrix, the sponsor resubmitted and received approval [46].

Table: CAR-T Cell Therapy Potency Assay Performance Characteristics

Assay Parameter	Binding Assay	Cytokine Release	Cytotoxicity Assay
Precision (%CV)	8.5%	12.3%	15.7%
Linearity (R²)	0.998	0.985	0.967
Range	1.56-100 ng/mL	3.1-200 pg/mL	1:1-1:16 E:T ratio
Stability-Indicating	Yes	Yes	Yes
MoA Relevance	Target recognition	T-cell activation	Biological function

Engineered Macrophage Therapy with Multimodal Mechanism

Challenge: Resolution Therapeutics developed a potency strategy for an engineered macrophage therapy with four distinct MoAs: phagocytosis, antigen presentation, inflammatory signaling, and tissue remodeling [18].

Solution: The team implemented a multi-attribute potency matrix that collectively reflected these diverse functions:

• Phagocytosis Assay: Quantified uptake of fluorescently-labeled tumor cells
• Surface Marker Expression: Monitored HLA-DR and co-stimulatory molecules
• Cytokine Secretion Profile: Measured IL-6, IL-10, IL-12, and TNF-α using multiplex platform
• Matrix Metalloproteinase Secretion: Assessed tissue remodeling capability

Regulatory Strategy: The company engaged with both FDA and EMA early in development, presenting the comprehensive matrix approach. Both agencies accepted the strategy with the FDA requiring additional validation data for the phagocytosis assay, while EMA focused on the overall control strategy and specifications for each attribute [18].

Diagram 2: Multimodal Potency Matrix for Engineered Cell Therapy. This diagram illustrates how multiple biological functions collectively contribute to the overall potency assessment for therapies with complex mechanisms of action.

The development of robust, MoA-linked potency assays remains a critical challenge in cell therapy development, with significant implications for regulatory approval across both US and EU jurisdictions. While regulatory expectations continue to evolve, the fundamental requirement remains constant: potency assays must reflect the biological activity that correlates with clinical effect.

The increasing acceptance of potency matrices for complex therapies represents a pragmatic approach to addressing multimodal mechanisms of action [18]. Meanwhile, regulatory harmonization efforts, particularly through ICH guidelines, may help align expectations between FDA and EMA, though regional differences in emphasis are likely to persist [2] [50].

For developers, early investment in understanding the MoA and developing quantitative, functional potency assays provides the strongest foundation for regulatory success. Implementing a phase-appropriate strategy that begins with qualified methods in early development and progresses to fully validated assays for commercialization remains the most effective approach for navigating the complex CMC landscape for cell therapies [48] [46].

As the field advances, emerging technologies including artificial intelligence, organoid-based systems, and advanced analytical platforms promise to enhance our ability to develop more predictive potency assays that accurately reflect clinical performance [49]. By maintaining a science-driven approach focused on biological relevance rather than mere compliance, developers can overcome the persistent challenges of potency assay development and bring transformative cell therapies to patients in need.

Ensuring Process Consistency with Highly Variable Autologous Starting Material

For developers of autologous cell therapies, where a patient's own cells are the starting material, achieving process consistency is a paramount challenge. Unlike traditional pharmaceuticals or allogeneic therapies from donor banks, autologous starting materials are inherently variable. This variability stems from factors such as the patient's disease status, age, prior treatments, and genetic background. The central thesis is that while the United States (US) and European Union (EU) regulatory frameworks share the ultimate goal of ensuring patient safety and product efficacy, their detailed Chemistry, Manufacturing, and Controls (CMC) requirements present distinct strategic considerations for navigating this variability. A deep understanding of both the divergences and convergences in the US and EU regulatory approaches is not merely an academic exercise but a critical component for the successful global development of these transformative medicines [2] [1].

This guide objectively compares the regulatory strategies required for the US and EU markets when dealing with variable autologous starting materials. It synthesizes current regulatory guidance and emerging best practices to provide a framework for developing robust, compliant, and effective manufacturing processes.

US and EU Regulatory Frameworks: A Comparative Analysis

The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) provide guidance for the development of cell therapies, known as Cell and Gene Therapies (CGTs) in the US and Advanced Therapy Medicinal Products (ATMPs) in the EU. A critical foundational difference lies in their classification systems. The EU possesses a more precise legal framework where a product containing genetically modified cells, such as many autologous CAR-T cells, is always classified as a Gene Therapy Medicinal Product (GTMP). In contrast, the US uses a broader umbrella category of "cellular and gene therapies" [1].

Table 1: Key Regulatory Differences for Autologous Therapies between the US and EU

Regulatory CMC Consideration	FDA (US) Position	EMA (EU) Position
Starting Material Definition	No formal regulatory definition; employs a risk-based approach for "critical raw materials." [2]	Legally defined; materials that become part of the drug substance must follow GMP principles. [2]
Donor Testing Requirements	Governed by 21 CFR 1271, typically performed in CLIA-accredited labs. [2]	Governed by the EU Tissue and Cells Directive (EUTCD), must be handled in licensed premises. Requires some testing even for autologous material. [2]
Facility GMP Expectations	A "fit-for-purpose" approach for Phase 1, focusing on safety. GMP rigor increases through phases. [1]	Requires GMP-grade manufacturing of investigational products for first-in-human studies. [1]
Process Validation (PV)	Number of batches not specified but must be statistically justified. [2]	Generally requires three consecutive batches, with some flexibility. [2]
Use of Surrogate Materials in PV	Allowed with sufficient justification. [2]	Allowed only in cases of a shortage of autologous starting material. [2]
Stability Data for Comparability	Prefers a thorough assessment, including real-time data for certain changes. [2]	Real-time data is not always mandatory; accelerated studies can be acceptable. [2]

Another significant operational difference involves importation. An autologous therapy manufactured in the US for use in an EU clinical trial requires the manufacturing site to have EU GMP certification and a Qualified Person (QP) to release the batch, even if the site is fully FDA-compliant [1].

Strategic Approaches to Process Control and Consistency

Navigating the variability of autologous starting material requires a multi-faceted strategy focused on control and understanding. The following experimental and control strategies are essential under both regulatory jurisdictions.

Comprehensive Incoming Material Characterization

The first line of defense against variability is a deep characterization of the incoming apheresis material. This goes beyond standard viability and cell count and should include:

• Phenotypic Profiling: Using high-resolution flow cytometry to determine the precise composition of T-cell and other immune cell subsets (e.g., CD4+/CD8+ ratio, memory phenotypes, exhaustion markers).
• Functional Potency Assays: Measuring the baseline functionality of the cells through assays for cytokine secretion, proliferation, or metabolic activity upon stimulation.
• Molecular Analysis: Assessing the genetic stability and potential impacts of prior therapies on the cell population.

This characterization data is not just for release; it is used to build Predictive Models that can inform process adjustments. For example, if an incoming batch has a low CD4+ T-cell count, a pre-defined protocol for adjusting culture conditions or media supplements can be initiated to ensure a consistent output.

Risk-Based Control of Critical Process Parameters (CPPs)

A risk-based approach to defining and controlling CPPs is a cornerstone of both FDA and EMA expectations [2]. The following diagram illustrates the logical workflow for identifying and controlling these parameters to ensure a consistent Critical Quality Attribute (CQA) profile in the final drug product.

The experimental protocol for this involves a Design of Experiments (DoE) approach. Instead of testing one factor at a time, a DoE systematically varies multiple CPPs (e.g., cell seeding density, cytokine concentrations, transduction multiplicity of infection, media exchange schedules) to model their individual and interactive effects on CQAs (e.g., vector copy number, transduction efficiency, final cell viability). This data-driven approach allows for the establishment of scientifically justified Proven Acceptable Ranges (PARs) for each CPP, which is far more robust than fixed setpoints and is viewed favorably by regulators.

Demonstrating Comparability After Process Changes

Making a change to the manufacturing process is inevitable. The FDA and EMA currently have nuanced differences in their guidance on demonstrating comparability for CGT products, as they are outside the scope of ICH Q5E, though a new annex is in development [2].

The FDA's draft guidance on "Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products" (July 2023) and the EMA's multidisciplinary guideline (effective July 2025) both advocate for a risk-based approach [2]. The extent of testing should be commensurate with the stage of development and the potential impact of the change. Both agencies agree that testing should include assessments of identity, purity, potency, and safety. However, the FDA often expects more functional potency assays and may place a greater emphasis on the inclusion of historical data in the comparability exercise, whereas the EMA's guidance for genetically modified cells outlines specific attributes to test when the manufacturing process for the viral vector (a starting material) is changed [2].

The Scientist's Toolkit: Essential Reagents and Materials

Success in managing autologous variability relies on a suite of specialized reagents and analytical tools. The following table details key solutions for process development and control.

Table 2: Key Research Reagent Solutions for Autologous Process Development

Reagent / Material	Function & Rationale
Cell Activation Reagents (e.g., anti-CD3/CD28 beads/antibodies)	Provides the essential primary signal for T-cell activation and proliferation, a critical step for subsequent genetic modification. Different reagent formats can impact activation kinetics and final cell phenotype.
High-Quality Cytokines (e.g., IL-2, IL-7, IL-15)	Supports T-cell survival, expansion, and can influence the differentiation of memory T-cell subsets, which is crucial for the persistence and potency of the final product.
Clinical-Grade Viral Vectors (e.g., Lentivirus, Gamma-retrovirus)	The vehicle for stable genetic modification (e.g., CAR gene insertion). The quality, titer, and purity of the vector batch are Critical Process Parameters directly impacting transduction efficiency and patient safety.
Serum-Free / Xeno-Free Culture Media	Provides a defined, consistent, and safe nutrient environment for cell growth. Eliminates lot-to-lot variability and immunogenic risks associated with serum, directly contributing to process consistency.
Advanced Flow Cytometry Panels	Enables high-resolution characterization of the starting material, in-process cells, and final product. Panels are designed to track critical quality attributes like immunophenotype, activation status, and expression of the transgene.
Molecular Biology Kits (ddPCR, qPCR, NGS)	Used for critical safety and identity tests, including vector copy number (VCN) analysis, detection of replication competent virus (RCV), and monitoring for off-target editing effects.

Analytical Methods and Orthogonal Testing

Regulators expect a comprehensive analytical package to demonstrate control over the process and product. Both the FDA and EMA have shown an openness to alternative or orthogonal methods where scientifically justified [1]. Orthogonal methods use different scientific principles to measure the same attribute, thereby building greater confidence in the result.

Table 3: Key Analytical Methods for Characterizing Autologous Cell Therapies

Quality Attribute	Primary Method	Orthogonal Method(s)	Regulatory Context
Potency	In vitro cytotoxicity assay against target cells.	Cytokine release assay (e.g., IFN-γ ELISA/Luminex); Transgene expression by flow cytometry.	The FDA emphasizes the need for a validated, biologically relevant functional potency assay by pivotal studies. This is a common CMC deficiency. [1]
Identity / Purity	Flow cytometry for cell surface markers (CD3, CD4, CD8) and CAR expression.	ddPCR for VCN; mRNA sequencing for TCR repertoire.	Used to confirm the product is the intended cell population and to quantify impurities.
Safety	Sterility tests (e.g., BacT/ALERT).	Mycoplasma testing, Endotoxin testing (LAL), and RCV testing.	FDA requires RCV testing on both the viral vector and the final cell product, whereas EMA may not require it on the final product if absence is shown on the vector. [2]
Genetic Stability	qPCR for VCN.	Next-Generation Sequencing (NGS) for integration site analysis.	NGS provides a much deeper characterization of the genetic modification, identifying the number of unique integration sites and potential risks from integration near oncogenes.

The following diagram illustrates a typical experimental workflow that integrates these analytical methods from the autologous starting material to the final cryopreserved product.

Ensuring process consistency with highly variable autologous starting materials is a complex but surmountable challenge. A successful global CMC strategy requires a deep understanding of the nuanced differences between the US and EU regulatory landscapes. While the EU has more formal definitions for starting materials and mandates GMP earlier, the FDA employs a phased, risk-based approach that escalates requirements with clinical progression. The convergence lies in the shared expectation for a science- and risk-based framework, robust process understanding, and comprehensive analytical comparability protocols. By implementing advanced characterization, controlled processes with defined PARs, and orthogonal analytical methods, developers can build the evidence needed to assure regulators in both regions that their autologous cell therapy is consistently safe, pure, and potent, despite the inherent variability of its origins.

For developers of cell and gene therapies, navigating the divergent Good Manufacturing Practice (GMP) compliance and inspection frameworks of the United States (US) and European Union (EU) presents a significant challenge. Chemistry, Manufacturing, and Controls (CMC) deficiencies account for approximately 74% of Complete Response Letters (CRLs) issued by the US Food and Drug Administration (FDA) for biological applications, highlighting the critical importance of robust manufacturing quality systems [4]. The European Medicines Agency (EMA) has similarly heightened expectations, with its new guideline on clinical-stage Advanced Therapy Medicinal Products (ATMPs) emphasizing that "immature quality development may compromise use of clinical trial data to support a marketing authorization" [14]. This comparative guide examines the distinct regulatory approaches to facility inspection and data integrity, providing drug development professionals with strategic insights for global compliance.

Comparative Analysis: US and EU GMP Compliance Frameworks

Fundamental Differences in GMP Philosophy and Implementation

The US FDA and EU EMA approach GMP compliance for cell therapies with fundamentally different verification systems and philosophical frameworks, particularly during clinical development phases [14].

Table: Comparative Analysis of US and EU GMP Compliance Requirements

Aspect	US FDA Approach	EU EMA Approach
GMP Application Timeline	Phase-appropriate application; full compliance verified at pre-license inspection [14]	Mandatory GMP compliance for all clinical trials, including early-phase studies [14]
Verification Mechanism	Reliance on sponsor attestation initially, with verification via FDA inspection at BLA stage [14]	Mandatory self-inspections with documented evidence of effective quality systems [14]
Facility Certification	Facility licensure as part of BLA approval [11]	Requires EU GMP certification and Qualified Person (QP) release for batches imported to Europe [1]
Viral Vector Classification	Classified as a biologic drug substance, requiring facility licensing and inspection [11]	Can be classified as starting materials, not always subject to same oversight level as drug substances [11]
Documentation System	Common Technical Document (CTD) format with "Drug Substance" and "Drug Product" terminology [14]	Common Technical Document (CTD) format with "Active Substance" and "Investigational Medicinal Product" terminology [14]

Inspection Readiness and Facility Requirements

The regulatory inspection frameworks differ substantially between regions, requiring tailored preparation strategies:

• EU Facility Requirements: The EU mandates that manufacturing sites possess EU GMP certification and have Qualified Persons (QPs) within the EU for batch release, regardless of FDA compliance status in the US [1]. This requirement extends to investigational products used in clinical trials, necessitating early planning for companies importing products to Europe.

• US Facility Requirements: The FDA classifies viral vectors used in cell therapy manufacturing as a drug substance, requiring facilities to be licensed and inspected for quality metrics related to vector purity, potency, safety, and handling [11]. The FDA's phase-appropriate approach allows for progressive implementation of GMP standards, with full compliance expected by the time of Biologics License Application (BLA) submission [14].

Data Integrity Requirements and Documentation Standards

Data Governance and Integrity Frameworks

Both regulatory authorities emphasize data integrity as a cornerstone of GMP compliance, with particular attention to electronic data systems and manufacturing records:

• Orthogonal Methods: Both FDA and EMA encourage using orthogonal assays (methods using different scientific principles to measure the same attribute) to build confidence in critical quality attributes (CQAs) [1]. For gene therapy programs, this typically requires at least two complementary methods for key attributes like vector genome integrity.

• Electronic Data Systems: The EU is updating its GMP guidelines through the revision of EudraLex Volume 4, Chapter 4, Annex 11, and the introduction of a new Annex 22 on Artificial Intelligence to address the implementation of AI systems in pharmaceutical manufacturing [51]. These updates aim to ensure clear, practical guidance for manufacturers applying digital technologies and AI in medicine production.

Analytical Method Validation

The approach to analytical method validation demonstrates both convergence and divergence in regulatory expectations:

• Phase-Appropriate Validation: The FDA applies a "phase-appropriate" lens to assay requirements, where early-phase (IND) assays need qualification but must be reliable, reproducible, and sensitive enough to support safety decisions [1]. By Phase 3 and into pre-registration, full validation is required under ICH Q2(R2).

• EMA's Evolving Position: EMA's guidelines for investigational ATMPs (effective July 2025) specifically state that orthogonal methods should be considered for analytical testing to ensure robustness and reliability of results, particularly when reference standards or validated methods are lacking [1]. For clinical trial materials, validated analytical methods are encouraged but not strictly required for early phases.

Table: Data Integrity and Documentation Requirements Comparison

Documentation Element	US FDA Expectations	EU EMA Expectations
Stability Data for Comparability	Thorough assessment including real-time data for certain changes [2]	Real-time data not always needed [2]
Historical Data Utilization	Inclusion of historical data recommended [2]	Comparison to historical data not required/recommended [2]
Process Validation Batches	Not specified, but must be statistically adequate based on variability [2]	Generally, three consecutive batches with some flexibility allowed [2]
Platform Data in Process Validation	Acceptable where same/similar manufacturing steps are used [2]	Acceptable where same/similar manufacturing steps are used [2]
Alternative Methodologies	Openness to accepting alternative methods where feasible and justifiable [1]	Clarified position on alternative methods, demonstrating increased commitment [1]

Inspection Preparedness: Protocols and Best Practices

Pre-Inspection Assessment Methodology

A comprehensive pre-inspection assessment should evaluate both US FDA and EU EMA requirements through a structured protocol:

Experimental Protocol 1: Facility Readiness Assessment

• Objective: Systematically evaluate compliance with both US and EU GMP requirements
• Methodology:
  • Conduct mock inspections using FDA and EMA/EU regulatory checklists
  • Verify data integrity through audit trails review for critical systems
  • Assess environmental monitoring data trends for cleanrooms
  • Review supplier qualification records for raw and starting materials
  • Verify training records for technical staff
• Acceptance Criteria: ≤5 major findings, complete documentation for all critical processes, successful data integrity audit

Experimental Protocol 2: Data Integrity Verification

• Objective: Ensure complete, consistent, and accurate data throughout product lifecycle
• Methodology:
  • Select representative batch records for traceability exercise
  • Verify ALCOA+ principles (Attributable, Legible, Contemporaneous, Original, Accurate, + Complete, Consistent, Enduring, Available) for critical data
  • Audit electronic system validation documentation
  • Review change control records for manufacturing processes
  • Assess data reconciliation between different systems
• Acceptance Criteria: 100% data traceability, no unresolved data discrepancies, validated electronic systems

Regulatory Inspection Workflow

The following diagram illustrates the coordinated approach to managing facility inspections under both regulatory frameworks:

Table: Research Reagent Solutions for GMP Compliance

Tool/Resource	Function	Application in GMP Context
Orthogonal Assays	Methods using different scientific principles to measure the same attribute [1]	Build confidence in critical quality attributes (CQAs); required for key measurements like vector genome integrity
Electronic Data Systems	Automated data capture and management systems	Ensure data integrity through audit trails; must comply with EU Annex 11 and FDA 21 CFR Part 11 requirements
Reference Standards	Qualified materials for assay calibration and validation	Essential for method validation; EDQM is introducing new primary labels with enhanced safety information [51]
Quality Management Software	Integrated systems for document control, CAPA, and change management	Centralize GMP documentation; facilitate inspection readiness through organized record-keeping
Environmental Monitoring Systems	Continuous monitoring of cleanroom conditions	Critical for aseptic processing; provides trend data for inspection review

Strategic Recommendations for Global Compliance

Achieving and maintaining facility and inspection readiness in both US and EU markets requires a strategic, integrated approach:

• Early Engagement: Sponsors should engage with both FDA CBER's Office of Therapeutic Products (OTP) and EMA's Committee for Advanced Therapies (CAT) early in development to align on expectations [35]. The FDA's INTERACT meetings and the EU's ATMP classification procedure provide opportunities for early alignment [1].

• Harmonized Quality Systems: Implement a unified quality system that accommodates both FDA's phase-appropriate approach and EU's full GMP requirements from clinical trials through commercialization. This includes building comprehensive data integrity protocols that satisfy both regulatory frameworks.

• Strategic Facility Planning: For global development, ensure manufacturing facilities can meet both FDA licensure requirements and EU GMP certification standards, including having Qualified Persons (QPs) for EU batch release [1].

• Comparability Strategy: Develop a robust comparability protocol for manufacturing changes, as both agencies require extensive testing to demonstrate equivalence after process modifications [2]. The FDA requires that the post-change product may no longer qualify for expedited programs like RMAT if comparability cannot be established [35].

By understanding these distinct regulatory frameworks and implementing comprehensive compliance strategies, cell therapy developers can enhance their inspection readiness and facilitate global market access for innovative therapies.

For cell and gene therapies (CGTs), the product stability and sterility assurance are not merely quality attributes but fundamental determinants of clinical success and regulatory approval. These advanced therapies, known as Advanced Therapy Medicinal Products (ATMPs) in the European Union (EU), present unique Chemistry, Manufacturing, and Controls (CMC) challenges due to their living nature and patient-specific design [11]. The complex logistics involved in maintaining product stability from manufacturing to patient infusion—often referred to as the "cold chain" or "cryochain"—require sophisticated coordination and monitoring systems [52]. Understanding the nuanced regulatory expectations between the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) is essential for developers aiming for global market access. This guide compares the specific technical requirements for shelf-life determination and cold chain management across these major regulatory jurisdictions, providing researchers and drug development professionals with actionable insights for compliance and operational excellence.

Regulatory Frameworks: US vs EU Comparison

Foundational Regulatory Structures

The regulatory categorization of cell therapies differs significantly between the US and EU, influencing subsequent requirements for stability and sterility. In the US, Cell and Gene Therapies (CGTs) are regulated as biologics by the FDA's Center for Biologics Evaluation and Research (CBER) [19] [11]. The EU employs the umbrella term Advanced Therapy Medicinal Products (ATMPs), which encompasses gene therapy medicines, somatic cell therapy medicines, and tissue-engineered products regulated by the EMA through its Committee for Advanced Therapies (CAT) [5] [11]. This fundamental distinction in classification often leads to differences in technical expectations, even when the underlying scientific principles remain consistent.

Recent Regulatory Developments

Both regulatory bodies have recently updated their guidance to address the unique challenges of cell therapy products:

• FDA: In September 2025, the FDA issued new draft guidance on "Expedited Programs for Regenerative Medicine Therapies for Serious Conditions," emphasizing that accelerated clinical development must not compromise CMC rigor, including stability testing and cold chain validation [35]. The FDA has also published draft guidance on "Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products" (July 2023) and "Potency Assurance for Cellular and Gene Therapy Products" (December 2023) [19].

• EMA: The EMA's "Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials" came into effect in July 2025, providing consolidated guidance on stability testing and cold chain management across over 40 previously separate documents [14].

The table below summarizes the key regulatory bodies and recent guidance documents relevant to stability and sterility of cell therapies:

Table 1: Regulatory Bodies and Guidance Documents for Cell Therapies

Aspect	US (FDA)	EU (EMA)
Primary Regulatory Center	Center for Biologics Evaluation and Research (CBER) [19]	Committee for Advanced Therapies (CAT) [5]
Recent Guidance on CMC	- Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products (Draft, July 2023) [19]- Potency Assurance for Cellular and Gene Therapy Products (Draft, December 2023) [19]	- Guideline on quality, non-clinical and clinical requirements for investigational ATMPs (Effective July 2025) [14]
Expedited Pathways	Regenerative Medicine Advanced Therapy (RMAT) Designation [35]	Priority Medicines (PRIME) Scheme [11]

Defining Shelf-Life and Stability

Stability Testing Requirements

Stability testing for cell therapies must demonstrate that the product maintains its critical quality attributes (CQAs) throughout its proposed shelf-life under defined storage conditions. Both regulators require real-time stability data, though with some differences in emphasis and approach.

• FDA Expectations: The FDA emphasizes phase-appropriate stability protocols, with increasing rigor through development phases [4]. For commercial approval, the agency expects comprehensive real-time stability data on consecutive batches representing the final manufacturing process [2]. The FDA has specifically highlighted potency assays as a common deficiency in CGT applications, requiring stability-indicating potency methods that are biologically relevant and functional [4].

• EMA Expectations: The EMA typically requires stability data from three consecutive batches for process validation, with some flexibility allowed for ATMPs addressing unmet needs [2]. The 2025 ATMP guideline stresses that "immature quality development may compromise use of clinical trial data to support a marketing authorization," indicating that inadequate stability data collected during trials could jeopardize approval [14].

Analytical Method Considerations

The validation of analytical methods used in stability testing follows similar scientific principles but differs in regulatory interpretation:

• Orthogonal Methods: Both agencies encourage orthogonal methods (using different scientific principles to measure the same attribute) to build confidence in stability data, particularly for critical attributes like identity, potency, and purity [1]. The FDA applies a phase-appropriate lens, requiring methods to be reliable and reproducible for early-phase studies, with full validation required by Phase 3 under ICH Q2(R2) [1].

• Potency Assays: Significant differences exist in potency testing expectations. For viral vectors used in vitro, the FDA expects validated functional potency assays to assess the efficacy of the drug product, while the EMA generally considers infectivity and transgene expression sufficient in early phases, with less emphasis on functional assays in later stages [2].

Table 2: Stability Testing Requirements Comparison

Stability Testing Aspect	US FDA Requirements	EU EMA Requirements
Batch Requirements	Not specified, but must be statistically adequate based on variability [2]	Generally three consecutive batches, with some flexibility [2]
Real-time Data for Comparability	Thorough assessment including real-time data for certain changes [2]	Real-time data not always needed [2]
Use of Historical Data	Inclusion of historical data recommended [2]	Comparison to historical data not required/recommended [2]
Potency Testing for Viral Vectors	Validated functional potency assay essential [2]	Infectivity and expression of transgene generally sufficient in early phase [2]
Stability-Indicating Methods	Functional, biologically relevant assays expected [4]	Orthogonal methods should be considered for robustness [1]

Cold Chain Logistics and Management

Temperature Requirements and Monitoring

Cell therapies typically require ultra-low temperature storage and transport, often at -150°C to -196°C, to maintain viability and potency [52]. Maintaining this "cryochain" presents significant logistical challenges, as any deviation can render the therapy non-viable [52]. Both regulators expect comprehensive temperature monitoring and control throughout the supply chain, though their specific expectations differ.

• FDA Perspective: The FDA emphasizes chain of custody and chain of identity maintenance throughout the cold chain, particularly for autologous products [52]. The agency expects temperature monitoring data to be available for review and that any excursions are thoroughly investigated for impact on product quality.

• EMA Perspective: The EU requires demonstration of compatibility between the medical device used in storage/transport and the cell therapy product, though the specific requirements are less detailed than FDA expectations [2]. The EMA's new ATMP guideline references the importance of cold chain maintenance but defers to specific technical guidelines on the topic [14].

Supply Chain Models

The complexity of cold chain logistics varies significantly based on the supply chain model employed:

• Centralized Manufacturing: The dominant model where therapies are produced in centralized GMP facilities and shipped to treatment centers [53]. This model benefits from economies of scale but faces challenges in maintaining the cryochain over long distances.

• Hybrid Models: Emerging approaches that combine centralized production with point-of-care (PoC) or decentralized manufacturing strategies [53]. These models reduce transport risks and time delays but require distributed quality systems.

• Point-of-Care Manufacturing: Fully decentralized manufacturing at the treatment site, eliminating complex transport logistics but requiring significant infrastructure investment and validation at multiple locations.

The diagram below illustrates the complex workflow and cold chain requirements for autologous cell therapies:

Diagram Title: Autologous Cell Therapy Cold Chain Workflow

Experimental Protocols for Stability and Sterility

Stability Study Design Protocol

Well-designed stability studies are essential for establishing scientifically justified shelf-life claims. The following protocol outlines a comprehensive approach:

• Sample Selection: Include a minimum of three consecutive batches manufactured at commercial scale [2]. For autologous therapies, include multiple donors representing expected biological variability.

• Storage Conditions: Implement real-time storage at the proposed long-term storage temperature (typically -150°C to -196°C for cryopreserved products) [52]. Include accelerated stability conditions where scientifically justified.

• Test Intervals: Schedule testing at minimum at 0, 3, 6, 9, 12, 18, and 24 months for real-time studies, with more frequent intervals for accelerated conditions.

• Test Parameters: Include all Critical Quality Attributes (CQAs) such as viability, potency, identity, purity, sterility, mycoplasma, endotoxin, and container closure integrity [2] [4].

• Stability-Indicating Methods: Employ validated, stability-indicating assays, particularly for potency, which should be biologically relevant and measure the mechanism of action [4].

Cold Chain Validation Protocol

Validating the entire cold chain ensures products maintain quality attributes during transport:

• Thermal Mapping: Perform comprehensive thermal mapping of all shipping containers under worst-case conditions, including different seasonal scenarios [52].

• Transport Simulation: Conduct simulated shipments using qualified equipment with continuous temperature monitoring at multiple points within the container.

• Excursion Studies: Define acceptable temperature excursion limits through supportive studies that expose products to controlled excursions with subsequent quality testing.

• Container Qualification: Qualify primary and secondary packaging under dynamic conditions that simulate vibration, pressure changes, and handling stresses [52].

The Scientist's Toolkit: Essential Research Reagents and Materials

The table below details essential materials and their functions in stability and sterility testing of cell therapies:

Table 3: Essential Research Reagents and Materials for Stability and Sterility Testing

Reagent/Material	Function	Key Considerations
Cryoprotectant Agents (CPAs)	Protect cells from ice crystal formation during freezing and thawing [52]	DMSO concentration (typically 5-10%); potential toxicity concerns; requires removal validation [52]
Liquid Nitrogen Storage Systems	Maintain ultra-low temperatures (-150°C to -196°C) for long-term storage [52]	Vapor phase vs. liquid phase storage; temperature monitoring and alarm systems; backup systems required
Cryoshippers	Transport systems maintaining cryogenic temperatures during transit [52]	Hold time validation; temperature monitoring capabilities; shipping validation under worst-case conditions
Cell Viability Assays	Measure cell survival and function post-thaw	Must use validated, stability-indicating methods; functional assays preferred over simple dye exclusion [4]
Sterility Testing Kits	Detect microbial contamination	Validation for product compatibility; rapid microbiological methods preferred for short shelf-life products
Mycoplasma Detection Kits	Detect mycoplasma contamination	PCR-based methods preferred for speed; requires validation for specific product matrix
Endotoxin Testing Kits	Detect bacterial endotoxins	Gel clot, turbidimetric, or chromogenic methods; validation required for product compatibility
Potency Assay Reagents	Measure biological activity	Must be biologically relevant and measure mechanism of action; requires extensive validation [4]

The regulatory landscapes for cell therapy stability and sterility in the US and EU, while founded on similar scientific principles, present distinct challenges in implementation. The FDA generally takes a more prescriptive approach to potency testing and method validation, while the EMA provides more specific guidance on batch requirements for stability studies. Both agencies are converging on the importance of orthogonal methods and risk-based approaches to stability testing and cold chain management. As regulatory frameworks evolve with the rapidly advancing science, developers should engage early with both agencies through available consultation pathways to align their stability and sterility strategies with regional expectations. The increasing adoption of digital monitoring technologies and hybrid supply chain models promises to enhance both compliance and patient access to these transformative therapies.

Side-by-Side Analysis: A Detailed Comparison of Key EU and US CMC Requirements

The development of cell and gene therapies, known as Advanced Therapy Medicinal Products (ATMPs) in the European Union (EU) and Cell and Gene Therapies (CGTs) in the United States, necessitates strict adherence to Good Manufacturing Practice (GMP) standards. The core difference in philosophy is foundational: the EU mandates full GMP compliance for medicines used in clinical trials, while the US employs a phase-appropriate GMP approach that evolves in stringency as a product moves through clinical development toward commercialization [1]. This distinction creates significantly different strategic and operational pathways for sponsors navigating these two major regulatory jurisdictions. Understanding these divergent paths is critical for global development plans, as the requirements impact everything from initial facility design and material sourcing to batch release and market authorization.

The regulatory frameworks themselves are structurally different. In the US, the Code of Federal Regulations, specifically 21 CFR 211, details GMP for finished pharmaceuticals, with the FDA's Center for Biologics Evaluation and Research (CBER) providing overarching guidance for CGTs [54] [19]. Conversely, the EU's system, detailed in EudraLex - Volume 4, is based on Union law that must be transposed into the national laws of its 27 Member States, potentially leading to nuanced interpretations in different countries [54]. For cell and gene therapy developers, this means that the EU is not a single monolithic entity from a regulatory perspective, while the US operates under a single federal standard.

Comparative Analysis of GMP Expectations

The following table summarizes the key differences in GMP expectations between the EU and US for cell and gene therapy clinical trials.

Table 1: Key GMP Expectation Differences for Clinical Trials

Aspect	European Union (EU)	United States (US)
Governance & Release	Mandatory Qualified Person (QP) certification for every batch released to the market [54] [55].	Batch release is the authority of the Quality Control Unit (21 CFR 211.22) [54].
Facility Requirements	A valid GMP certificate for the manufacturing site is required, issued by a national competent authority following an inspection [55].	Facility must be "fit-for-purpose" for Phase 1, with emphasis on patient safety and sterility [1].
Starting Material & Ancillary Materials	Requires GMP-grade manufacturing of investigational products for first-in-human studies [1].	Expectations are "phase-appropriate," starting with higher-quality inputs than for small molecules [1].
Approach Philosophy	Mandate-based: Full GMP compliance is required from the start of clinical trials [55].	Phase-Appropriate: GMP stringency increases through clinical phases, from "fit-for-purpose" to full validation [1].
Importation for Trials	Requires an import license from the regulatory authority of the Member State where the product is imported [55].	Governed by FDA regulations for investigational products; no equivalent import license from a national authority.
CMC Focus	Focus on full GMP compliance from the outset.	Early phase focus on patient safety and sterility; process consistency in Phase 2; fully validated, commercial-ready processes by Phase 3 [1].

The Qualified Person (QP) vs. Quality Control Unit

A distinct and critical difference in batch release exists between the two regions. In the EU, the Qualified Person (QP) is a legally mandated individual who must certify that every batch of a medicinal product for a clinical trial or the market has been manufactured and tested in full compliance with EU GMP directives, the requirements of the Investigational Medicinal Product Dossier (IMPD), and the marketing authorization [54] [55]. The QP can be a member of the quality unit or an independent consultant, but their certification is a statutory requirement that cannot be waived [54]. This release is a two-step process, involving both QP certification and a separate regulatory release by the sponsor [55].

In contrast, the US system vests release authority in the Quality Control Unit, as defined in 21 CFR 211.22 [54]. This is a functional unit within the company's structure, not a specific legally designated individual like the QP. The unit is responsible for approving or rejecting all components, drug product containers, closures, in-process materials, packaging materials, labeling, and drug products. The responsibility is institutional, and the approach to fulfilling these responsibilities is expected to become more rigorous as the product advances through clinical phases toward a Biologics License Application (BLA).

Facility & Import Controls: GMP Certificates and Licenses

The EU requires a formal GMP certificate for the manufacturing site of the investigational product [55]. This certificate is issued by a national competent authority (e.g., the MHRA in the UK or a regulatory body in an EU Member State) following a successful GMP inspection. These certificates are entered into the publicly available EudraGMDP database, and their authenticity is mutually recognized across the EU [55]. This provides a transparent record of a site's GMP compliance status.

Furthermore, if an investigational product is manufactured outside the EU, an import license is required from the regulatory authority of the specific Member State into which the product is being imported [55]. The entire supply chain, including transportation and storage, must adhere to Good Distribution Practice (GDP) guidelines to maintain product quality. The sponsor holds ultimate responsibility for ensuring this controlled chain of distribution [55].

The US FDA does not issue an equivalent GMP certificate or import license from a national authority for clinical trial materials. Instead, the FDA conducts its own inspections of domestic and foreign manufacturing facilities to assess compliance with current GMP (cGMP) regulations [55].

Experimental Protocols and Methodologies

Protocol for EU QP Certification and Batch Release

The process for certifying and releasing a batch of an investigational cell or gene therapy for use in an EU clinical trial is a rigorous, document-heavy process. The methodology is designed to provide absolute traceability and quality assurance from starting material to patient.

Table 2: Key Reagents for QP Certification and Batch Release

Research Reagent / Document	Function in the Protocol
Product Specification File (PSF)	A comprehensive master file containing all documentation needed by the QP to certify a batch, including specifications, manufacturing records, and test results [55].
Investigational Medicinal Product Dossier (IMPD)	The central regulatory document detailing the product's quality, manufacture, and control, which the QP uses as a reference for certification [55].
Certificate of Analysis (CoA)	Provides certified test results for a specific batch of a raw material, ancillary material, or the final drug product, proving it meets pre-defined specifications [56].
Drug Master File (DMF)	A confidential, detailed document submitted to the FDA (and potentially referenced for EU) that provides information on the chemistry, manufacturing, and controls of a drug substance or material [56].
Technical Agreement	A contract that clearly defines the responsibilities of all parties involved in the supply chain (sponsor, manufacturer, importer, QP) [55].

Workflow Description: The process begins well before manufacturing, with the sponsor identifying a suitable QP 6-9 months prior to the clinical trial application submission [55]. The QP must be granted access to all documents related to the supply chain to schedule necessary GMP audits and evaluate the status of all sites involved. The foundational document, the Product Specification File (PSF), is developed with the QP's input. After a batch is manufactured, the QP reviews the entire batch record, all quality control testing data (e.g., sterility, potency, identity), and supporting documentation against the approved IMPD. Only upon verifying full compliance does the QP provide their certification. This is followed by the sponsor's regulatory release, confirming that all clinical trial authorization conditions are met.

Protocol for US Phase-Appropriate CMC Development

The US phase-appropriate approach is an iterative validation process where Chemistry, Manufacturing, and Controls (CMC) strategies evolve in parallel with clinical development. The methodology is based on risk management and scientific justification, with the bar for evidence raising at each phase.

Workflow Description: The process is segmented into three main clinical phases. In Phase 1, the primary focus is on patient safety. The manufacturing facility must be "fit-for-purpose," with the emphasis on ensuring sterility and product safety. The analytical methods used need to be qualified—demonstrated as reliable and reproducible—but not yet fully validated. By Phase 2, the focus shifts to demonstrating process consistency. Critical Process Parameters (CPPs) and Critical Quality Attributes (CQAs) must be identified and refined. Specifications for the product are tightened, and phase-appropriate validation begins. Finally, in Phase 3, the sponsor must demonstrate a fully GMP-compliant and validated process that is ready for commercial supply. All analytical methods must be fully validated according to ICH guidelines, and the quality of all materials must be at the commercial grade [1].

Analysis of Material Controls

Control of Ancillary and Starting Materials

Control over raw materials, particularly those of biological origin, is a critical point of convergence and slight divergence between the EU and US systems. Both regions enforce stringent requirements, but the EU's stance is often perceived as more prescriptive from the onset.

Internationally, guidelines like USP <1043> and reports from the International Pharmaceutical Regulators Programme (IPRP) provide a framework for categorizing and controlling ancillary materials based on risk assessment [56]. There is a strong emphasis on supplier quality agreements, Certificates of Analysis (CoA), and for materials of animal or human origin, detailed documentation proving the material has been purified and tested for adventitious agents [56]. The European Pharmacopoeia (5.2.12) further mandates that raw materials be produced under a suitable quality management system, with specific requirements for sterility, biological activity, and the control of impurities [56].

A key difference emerges for advanced therapies. The US FDA expects "higher quality input materials" than for early-phase small molecules and does not allow research-grade excipients or starting materials [1]. Meanwhile, the EMA's updated guideline emphasizes that ex vivo genome editing machinery must be defined as starting materials, not raw materials, requiring their manufacturing to adhere to GMP principles [1]. This classification has significant implications for developers, as it brings more of the production process under the umbrella of strict GMP compliance earlier in development within the EU.

The choice between the EU's mandated GMP pathway and the US's phase-appropriate approach is not one of right or wrong, but of strategic alignment with a product's development timeline and a company's resources. The EU system prioritizes a high level of quality assurance from the very first human dose, enforced through legally binding roles like the Qualified Person and system-wide controls like GMP certificates. This can de-risk patient safety but imposes significant upfront costs and operational hurdles. The US system offers a more graduated pathway, allowing CMC development to mature alongside clinical evidence, which can be particularly advantageous for small biotechs and academic innovators with limited initial funding.

For global developers, the imperative is to recognize these differences early. A successful international strategy must account for the EU's need for a QP, GMP-certified sites, and import licenses from the outset, while simultaneously planning for the FDA's escalating CMC expectations through Phases 1, 2, and 3. Proactive engagement with regulators via meetings like the FDA's INTERACT or pre-IND and the EMA's CAT classification procedure is essential to navigate this complex landscape and ensure that these transformative therapies can reach patients in both regions efficiently and safely.

For developers of cell and gene therapies (CGTs), known as advanced therapy medicinal products (ATMPs) in the European Union, demonstrating product potency through robust analytical testing presents a significant regulatory challenge with notable divergences between U.S. and EU requirements [2] [1]. Potency assays, defined as in vitro bioassays that measure the biological activity of a product, are critical release tests that confirm a therapy can achieve its intended biological effect [57] [43]. These assays serve multiple essential functions throughout the product lifecycle: they ensure manufacturing consistency, support stability studies, and provide crucial data for comparability exercises following process changes [58] [57].

The regulatory landscape for these complex biologics is evolving rapidly, with recent guidelines including the EMA's multidisciplinary guideline on investigational ATMPs that came into effect in July 2025 [14]. While both the FDA and EMA require potency assays that reflect the mechanism of action (MoA), their specific expectations for viral vector characterization and functional assay validation demonstrate key differences that sponsors must navigate for successful global development programs [2] [57].

Comparative Analysis of US and EU Regulatory Expectations

Fundamental Divergences in Testing Philosophies

The FDA and EMA approach potency verification with different emphases, particularly regarding the stage at which fully validated, MoA-reflective assays are required and the acceptance of surrogate methods [2] [57]. The FDA generally expects functional potency assays earlier in development, emphasizing biological relevance, while the EMA may initially accept simpler, physicochemical methods provided they correlate with biological activity [2].

Table 1: Comparative Overview of FDA and EMA Potency Testing Expectations

Regulatory Aspect	U.S. FDA Position	EU EMA Position
Potency testing for viral vectors for in vitro use	Validated functional potency assay essential to assess efficacy of drug product used in pivotal studies [2]	Infectivity and expression of transgene generally sufficient in early phase with less functional assays acceptable at later stages [2]
Assay validation timing	Phase-appropriate approach; assays must be reliable and reproducible for Phase 1, full validation required by Phase 3 under ICH Q2(R2) [1]	Validated analytical methods encouraged but not strictly required for early phases; orthogonal testing bolsters confidence [1]
Use of orthogonal methods	Generally encouraged to build confidence in critical quality attributes (CQAs); expected for identity, potency, and purity [1]	Specifically recommended in analytical testing to ensure robustness when reference standards are lacking [1]
Potency assay requirements for lot release	MoA-reflective functional assays mandated for lot release [57]	May accept surrogate assays under specific conditions [57]
Stability data for comparability	Thorough assessment including real-time data for certain changes [2]	Real-time data not always needed [2]

Viral Vector Classification and Testing Implications

A fundamental regulatory difference affecting potency strategy lies in how viral vectors are classified. The FDA classifies in vitro viral vectors used to modify cell therapy products as a drug substance, requiring full characterization and control [2]. In contrast, the EMA may classify these same vectors as starting materials, potentially reducing direct regulatory scrutiny but still requiring demonstration of quality [2] [11].

This classification difference creates a ripple effect throughout the testing strategy. For FDA submissions, viral vectors must undergo rigorous potency verification with validated functional assays, while EMA submissions may focus more on infectivity and transgene expression [2]. Additionally, testing for replication competent virus (RCV) differs: the EMA may not require further RCV testing on the final cell-based product if absence was demonstrated on the vector, whereas the FDA requires testing of both the vector and the final cell product [2].

Analytical Methodologies and Experimental Approaches

Established Potency Testing Strategies for Approved Products

Analysis of FDA-approved cell therapy products reveals practical insights into successful potency strategies. A review of 31 approved CTPs identified 104 total potency tests, averaging 3.4 tests per product [43]. These tests broadly fall into five categories, with viability/cell count and expression profiling being most frequently employed together.

Table 2: Potency Test Categories for FDA-Approved Cell Therapy Products

Test Category	Frequency (%)	Examples	Common Applications
Viability and Count	52%	Total nucleated cells, viable CD34+ cell count [43]	Cord blood products, cellular integrity
Expression	27%	CAR expression by flow cytometry, transgene expression [43]	Genetically modified products, surface markers
Bioassays	7%	IFN-γ release, cytotoxic activity [43]	CAR-T cells, TCR-modified cells
Genetic Modification	9%	Vector copy number (qPCR), percent LVV+ cells [43]	Gene-modified autologous therapies
Histology	3%	Tissue organization, viability retention [43]	Tissue-engineered products

For gene-modified products like CAR-T cells, the most common potency strategy combines CAR expression quantification (e.g., by flow cytometry) with functional bioassays measuring cytokine release (e.g., IFN-γ) upon antigen stimulation [43]. This orthogonal approach addresses both quantitative and qualitative aspects of potency, satisfying regulatory expectations for MoA reflection.

Experimental Protocols for Critical Potency Assays

Cytokine Release Bioassay for CAR-T Products

Purpose: To measure T-cell activation and functionality through interferon-gamma (IFN-γ) production upon engagement with target antigens [43].

Workflow:

• Cell Preparation: Harvest and count CAR-T cells, ensuring viability >70%
• Antigen Exposure: Co-culture CAR-T cells with CD19-expressing target cells (e.g., NALM-6 cells) at effector:target ratios of 1:1 to 10:1
• Incubation: Culture for 18-24 hours at 37°C, 5% CO₂
• Supernatant Collection: Centrifuge plates and collect supernatant
• IFN-γ Quantification: Measure IFN-γ concentration using ELISA or multiplex immunoassay
• Data Analysis: Calculate potency relative to reference standard, typically using parallel-line analysis

Validation Parameters: According to ICH Q2(R2), demonstrate accuracy, precision (repeatability and intermediate precision), specificity, linearity, range, and robustness [1] [57].

Vector Potency Assay for Gene Therapy Products

Purpose: To assess the functional transduction efficiency and transgene expression capability of viral vectors [2] [58].

Workflow:

• Cell Line Preparation: Culture permissive cell line (e.g., HEK293) to 70-80% confluence
• Vector Serial Dilution: Prepare 3-5 fold serial dilutions of viral vector reference and test samples
• Transduction: Infect cells with vector dilutions using appropriate multiplicity of infection (MOI)
• Expression Period: Incubate for 48-72 hours to allow transgene expression
• Detection: Quantify transgene expression using flow cytometry (for reporter genes) or quantitative PCR
• Potency Calculation: Determine relative potency by comparing test sample to reference standard using parallel-line analysis

Critical Reagents: Qualified cell banks, reference standard, detection antibodies, and appropriate controls are essential for assay performance [57].

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful potency assay development requires carefully selected and qualified reagents. The following table outlines critical materials and their functions in establishing robust analytical methods.

Table 3: Essential Research Reagents for Potency Assay Development

Reagent Category	Specific Examples	Function in Potency Testing	Qualification Requirements
Cell-Based Systems	Reporter cell lines, antigen-presenting cells [57]	Provide biological context for functional assays; express target antigens	Authentication, stability, passage number limits, mycoplasma testing
Reference Standards	Characterized drug substance, biological reference materials [57]	Serve as comparators for potency calculations; ensure assay continuity	Comprehensive characterization, stability data, establishment of acceptance criteria
Detection Reagents	Fluorochrome-labeled antibodies, ELISA kits [43]	Enable quantification of specific analytes or cell surface markers	Specificity testing, titration to determine optimal concentration
Critical Assay Components	Culture media, serum, cytokines [57]	Support cell growth and function during assay execution	Testing for performance, endotoxin levels, lot-to-lot consistency
Vector-Specific Reagents	Viral vectors, transduction enhancers [58]	Enable genetic modification of target cells for functional assessment	Titration, sterility, identity testing

Navigating Global Development: Strategic Considerations

Harmonization Opportunities in Comparative Testing

Despite regulatory differences, sponsors can identify strategic harmonization opportunities. Both agencies acknowledge the value of orthogonal methods that measure the same attribute through different scientific principles [1]. Additionally, a risk-based approach to comparability exercises is recognized by both FDA and EMA, where the extent of testing is proportionate to the stage of development and potential impact of manufacturing changes [58].

The emerging ICH Q5E annex specifically addressing CGT comparability may further align expectations between regions [2]. Currently, both agencies agree that the extent of testing should increase with the stage of clinical development, and that characterization tests should be added as appropriate [2].

Practical Implementation Roadmap

For sponsors navigating these complex requirements, a phased approach to potency assay development is recommended:

• Early Phase (Phase 1): Focus on patient safety and sterility assurance with qualified, reliable assays [1]. Implement orthogonal methods where feasible to build confidence in critical quality attributes [1].

• Mid-Stage Development (Phase 2): Begin refining critical process parameters and tightening specifications [1]. Transition toward more MoA-reflective functional assays, particularly for FDA submissions [2].

• Late Stage (Phase 3): Implement fully validated, GMP-compliant potency assays suitable for commercial supply [1]. For FDA, ensure validated functional potency assays are in place; for EMA, demonstrate correlation between surrogate and functional methods if applicable [2] [57].

Engaging with regulators through early consultation opportunities (such as the FDA's INTERACT meetings or EU national competent authority advice procedures) can help resolve classification uncertainties and align potency strategies with regional expectations [1].

The divergent expectations for potency and analytical testing of viral vectors and functional assays between US and EU regulators reflect philosophical differences in regulatory approach, yet both share the common goal of ensuring product safety and efficacy. Successful global development requires understanding these nuances while identifying harmonization opportunities.

As the regulatory landscape evolves with new guidelines and emerging technologies, the fundamental principles of developing MoA-reflective, validated potency assays remain constant. By implementing strategic, phase-appropriate approaches and engaging early with regulatory agencies, sponsors can navigate these complex requirements and advance promising cell and gene therapies to patients in both major markets.

For researchers and drug development professionals in the field of cell therapy, navigating the regulatory landscape for donor testing is a critical component of Chemistry, Manufacturing, and Controls (CMC). The donor eligibility criteria and associated testing requirements form the first defensive barrier against the introduction, transmission, and spread of communicable diseases through human cells, tissues, and cellular and tissue-based products (HCT/Ps) [59]. This guide provides a detailed, objective comparison of the donor testing frameworks established in the United States (US) under 21 CFR Part 1271 and the European Union (EU) under the Tissues and Cells Directives (EUTCD), synthesizing information from regulatory documents and comparative analytical studies to support robust global development strategies [60] [61].

Regulatory Framework and Definitions

The regulatory approaches in the US and EU, while aiming for a similar goal of patient safety, are built upon distinct legal foundations and product classifications, which subsequently influence donor testing obligations.

• United States (21 CFR 1271): The US Food and Drug Administration (FDA) regulates HCT/Ps under a unified framework codified in 21 CFR Part 1271 [59]. This regulation is primarily derived from the authority of Section 361 of the Public Health Service (PHS) Act, which focuses on preventing the spread of communicable diseases. The definition of HCT/Ps is broad, covering "articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient" [59]. A critical distinction is made between products regulated solely under Section 361 (which includes many minimally manipulated products) and those regulated as drugs, devices, and/or biological products under Section 351 of the PHS Act and the Federal Food, Drug, and Cosmetic Act. Donor testing requirements are a cornerstone for all HCT/Ps, regardless of their regulatory pathway [59].

• European Union (EUTCD): In the EU, Advanced Therapy Medicinal Products (ATMPs), which include gene therapies, somatic cell therapies, and tissue-engineered products, are regulated under the centralized medicinal product framework [62]. However, the starting point for donor eligibility and testing is the European Union Tissues and Cells Directives (EUTCD) [2]. These directives set the standards for the quality and safety of human tissues and cells intended for human application, which then feed into the requirements for the final medicinal product. The regulatory responsibility is shared, with the European Medicines Agency (EMA) evaluating marketing authorizations for ATMPs, but the donor testing standards are heavily influenced by the EUTCD [62] [2].

Comparative Analysis of Key Donor Testing Provisions

The following section breaks down the specific requirements for donor testing, highlighting both convergent and divergent expectations between the two regulatory systems.

Core Testing Requirements for Communicable Diseases

Both regions mandate testing for a defined set of communicable disease agents, but the specific pathogens and applicability can differ. The US regulations provide a highly detailed and prescriptive list.

Table 1: US 21 CFR 1271 Core Communicable Disease Agent Testing Requirements [59]

Disease Agent/Disease	Required For
Human Immunodeficiency Virus, types 1 and 2 (HIV-1/2)	All HCT/Ps
Hepatitis B Virus (HBV)	All HCT/Ps
Hepatitis C Virus (HCV)	All HCT/Ps
Human Transmissible Spongiform Encephalopathy (TSE), including Creutzfeldt-Jakob Disease	All HCT/Ps
Treponema pallidum (Syphilis)	All HCT/Ps
Human T-lymphotropic virus, type I (HTLV-I)	Viable, leukocyte-rich HCT/Ps
Human T-lymphotropic virus, type II (HTLV-II)	Viable, leukocyte-rich HCT/Ps
Chlamydia trachomatis	Reproductive HCT/Ps
Neisseria gonorrhea	Reproductive HCT/Ps

The US regulations also include a provision for "relevant communicable disease agents or diseases" not explicitly listed, if they meet specific criteria for risk, severity, and the availability of a suitable screening test [59]. While the EU's EUTCD also mandates testing for HIV, HBV, HCV, and Syphilis, a key operational difference lies in the requirement for autologous donors. The EMA requires some donor testing even for autologous material, whereas the FDA's requirements for autologous donors are less extensive or may be subject to exceptions [2].

Donor Screening and Eligibility Determination

Beyond analytical testing, both regions require a comprehensive assessment of the donor's medical history and behavioral risk.

• United States: The FDA requires a "donor medical history interview" [59]. This is a documented dialogue about the donor's medical history and relevant social behavior. For deceased donors, a "physical assessment" is required to check for signs of communicable disease. The determination of donor eligibility must be based on results from screening and testing, and the HCT/P must be accompanied by these eligibility records [59].

• European Union: Similarly, the EUTCD requires a detailed donor characterisation, which includes a health and lifestyle assessment, a medical history review, and a physical examination where appropriate. The objective is to identify risk factors that may make the donor unsuitable, thereby ensuring the safety of the recipients [2].

Laboratory and Quality System Requirements

The operational environment in which testing is performed is another area of nuanced difference.

• United States: FDA regulations stipulate that testing must be performed by a laboratory that is certified under the Clinical Laboratory Improvement Amendments (CLIA) or has met equivalent requirements, as deemed by the FDA [59]. This ensures a baseline standard for laboratory testing quality and reliability.

• European Union: The EUTCD requires that tissues and cells be handled and tested in "licensed premises and accredited centres" [2]. This creates a similar framework of oversight, though the specific accreditation bodies and processes are specific to the EU member states.

Experimental Protocols and Methodologies

This section outlines the standard experimental workflows for establishing donor eligibility under both regulatory frameworks. Adherence to these validated protocols is essential for generating reliable and regulatory-compliant data.

Protocol 1: Donor Eligibility Determination Workflow (US 21 CFR 1271)

Objective: To determine the eligibility of a donor of HCT/Ps based on required screening and testing to prevent the transmission of communicable diseases [59].

Methodology:

• Donor Identification and Consent: Obtain positive identification of the donor and acquire informed consent for screening and testing.
• Donor Medical History Interview (DMHI): Conduct a documented interview with the living donor or an appropriate representative for a deceased donor. Inquire about medical history, social behaviors, and travel history that may indicate a risk for communicable diseases.
• Physical Assessment and Review of Medical Records: For deceased donors, perform a physical assessment (limited autopsy or recent pre/postmortem exam) for signs of communicable disease. Review all available relevant medical records.
• Blood Sample Collection: Collect a blood sample from the donor using an approved method. For deceased donors, collect a sample within 24 hours of death or cardiopulmonary arrest.
• Communicable Disease Testing: Test the donor sample using FDA-licensed, approved, or cleared tests for the required agents listed in §1271.85 (e.g., HIV, HBV, HCV, etc.). Testing must be performed in a CLIA-certified lab or equivalent.
• Eligibility Determination: A designated responsible person must review all information (DMHI, physical assessment, medical records, test results) to determine eligibility. The donor is eligible only if all requirements are met.
• Documentation and Record Keeping: Create and maintain a record of the donor eligibility determination. The HCT/P from an eligible donor must be clearly labeled. HCT/Ps from ineligible donors must be quarantined and disposed of, or used only under urgent medical need with specific documentation.

Protocol 2: Donor Characterisation and Testing Workflow (EU Tissues and Cells Directives)

Objective: To ensure the quality and safety of human tissues and cells for human application through rigorous donor characterisation and testing in accordance with the EUTCD [2].

Methodology:

• Donor Selection and Informed Consent: Select donors based on initial selection criteria and obtain documented informed consent.
• Donor Characterisation (Health and Lifestyle Evaluation): Perform a comprehensive health and lifestyle assessment of the donor through a structured questionnaire and interview. This assessment aims to identify risk factors and exposures that could lead to transmissible diseases.
• Medical History and Physical Examination: Conduct a review of the donor's medical history and, where applicable, a physical examination to identify any contra-indications to donation.
• Biological Sample Collection: Collect appropriate biological samples (e.g., blood) for testing at the time of donation or, for living donors, within 7 days prior to procurement.
• Laboratory Testing: Test the donor samples for the required infectious markers (e.g., HIV, HBV, HCV, Syphilis) in a licensed and accredited laboratory. The EUTCD specifically requires testing for autologous donors, a key difference from the US approach.
• Assessment of Suitability: A qualified person (e.g., a physician) assesses all collected data (characterisation, medical history, test results) to make a final determination on the donor's suitability.
• Traceability and Record Management: Ensure full traceability of the donor and all donated material. Maintain all records related to donor characterisation and testing for the required archival period.

The following workflow diagram illustrates the key procedural similarities and the critical point of divergence in autologous donor testing between the two regulatory frameworks.

Figure 1: Comparative Workflow for Donor Eligibility and Suitability Determination. A key procedural difference is the mandatory testing of autologous donors in the EU, whereas US requirements for autologous donors are less extensive or subject to exceptions.

The Scientist's Toolkit: Research Reagent Solutions

Navigating donor testing requirements necessitates the use of specific, validated reagents and materials. The following table details key components essential for compliance with both US and EU standards.

Table 2: Essential Research Reagents and Materials for Donor Testing

Item	Function in Donor Testing	Key Regulatory Consideration
FDA-licensed/CE-marked Serological Assays	Detect specific antibodies against HIV, HBV, HCV, etc., indicating exposure.	Tests must be licensed/approved by the respective authority (FDA in US, CE-marked in EU) for donor screening [59].
FDA-licensed/CE-marked Nucleic Acid Testing (NAT) Kits	Directly detect viral genetic material (e.g., HIV RNA, HBV DNA), reducing the "window period" of infection.	NAT is often required for enhanced sensitivity. Kits must be appropriately validated and licensed [59].
Donor Screening Questionnaire	Standardized tool for conducting the donor medical history interview (US) or health and lifestyle assessment (EU).	Must be comprehensive, covering travel, medical, and behavioral history as per regulatory guidance [59] [2].
Validated Sample Collection Kits	Ensure sterile and standardized collection of blood or tissue samples from donors for testing.	Kits must prevent contamination and sample mix-ups, supporting data integrity and traceability.
Documentation and Record Management System	Tracks donor identity, test results, eligibility determination, and final disposition of the HCT/P.	Critical for audit trails and demonstrating compliance with Good Tissue Practice (GTP) and EUTCD traceability requirements [59] [2].

The donor testing requirements of the US 21 CFR 1271 and the EU Tissues and Cells Directives share a common foundation in their goal to ensure patient safety. Both mandate rigorous donor screening, specific communicable disease testing, and robust quality systems. The most significant operational difference lies in the mandatory testing of autologous donors in the EU, a requirement not uniformly applied in the US [2]. Furthermore, while both regions require high-quality laboratory testing, the specific oversight frameworks differ (CLIA certification in the US vs. accreditation and licensing in the EU) [59] [2]. For drug development professionals, understanding these nuanced similarities and differences is not merely an exercise in regulatory compliance but a critical CMC activity essential for designing efficient global development and marketing authorization strategies for cell-based therapies.

For cell and gene therapies (CGTs), known as Advanced Therapy Medicinal Products (ATMPs) in the European Union, demonstrating that a manufacturing process consistently produces a product meeting its predefined quality attributes is a critical regulatory requirement. Process validation provides this assurance and is a cornerstone of Chemistry, Manufacturing, and Controls (CMC). A central and historically rooted question in validation strategy is whether to use a fixed number of consecutive batches or a statistically justified number of batches. This guide objectively compares these two approaches within the context of evolving US and EU regulatory frameworks for CGTs.

The regulatory landscape for these complex biologics is dynamic. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) offer guidance on process validation, yet their perspectives show nuanced differences. Furthermore, a global trend is shifting the focus from traditional, fixed-number approaches toward a more risk-based, scientific, and data-driven lifecycle paradigm. Understanding these distinctions is crucial for developers aiming for global market approval, as CMC issues account for a significant percentage of regulatory delays and complete response letters [4].

Regulatory Landscape: US vs. EU Comparison

The FDA and EMA align on core principles but differ in specific expectations for process validation, particularly in the number of batches required for Process Performance Qualification (PPQ).

The following table summarizes the official positions of the two major regulatory bodies.

Table 1: Regulatory Positions on Batch Numbers for Process Validation

Regulatory Body	Official Position on Batch Numbers	Key Guidance Documents	Underlying Principle
US FDA	No mandated number. The number of batches must be statistically adequate based on process variability and risk [2] [17].	Process Validation: General Principles and Practices (2011)	Scientific, risk-based, and data-driven justification. The focus is on process understanding and control.
EU EMA	Generally three consecutive batches. Some flexibility is allowed, particularly for ATMPs addressing unmet medical needs [2].	EU Annex 15: Qualification and Validation (2015)	A traditionally accepted standard that provides a baseline for demonstrating consistency, with emerging flexibility.

Detailed Analysis of Regulatory Views

• US FDA Perspective: The FDA's guidance is clear that the number of batches must be "statistically adequate based on variability" [2] [17]. This means that for a highly variable and complex process, such as the manufacturing of a cell therapy, more than three batches may be required to provide sufficient confidence that the process is in a state of control. The agency emphasizes a lifecycle approach, integrating process design, qualification, and continued verification [63].
• EU EMA Perspective: The EMA's stance, as outlined in Annex 15, generally expects three consecutive batches for process validation [2]. However, this is not an absolute rule. There is a recognized flexibility, especially for ATMPs under programs like PRIME (Priority Medicines) or those addressing an unmet medical need, where concurrent validation may be permitted [2]. The "three-batch" rule is deeply ingrained in the industry standard for the EU.

The "Three Consecutive Batches" Paradigm: Rationale and Workflow

The use of three consecutive batches for process validation is a historical industry standard that persists due to its practical and statistical rationale.

Historical and Statistical Justification

The logic behind three batches is rooted in basic statistical reasoning and a rule of thumb for demonstrating consistency:

• First Batch Success: May be considered an accidental or fortuitous result [64] [65].
• Second Batch Success: Suggests the result is regular, but two data points can only define a straight line and are insufficient to assess variation or control robustly [64].
• Third Batch Success: Provides the minimum number of data points to begin evaluating variation and provides a higher level of confidence that the process is reproducible and under control, thus achieving validation [64] [65].

This approach became a baseline because it offers a defensible, though minimal, demonstration of consistency while balancing the high cost and time constraints of manufacturing additional validation batches, which is especially pertinent for costly CGTs [64].

Traditional Validation Workflow

The following diagram illustrates the typical workflow and decision logic within the traditional three-batch validation paradigm.

Diagram 1: Traditional Three-Batch Validation Workflow. This linear "pass/fail" approach requires three consecutive successful batches to deem a process validated.

The Statistically Justified Approach: A Risk-Based Framework

The modern, statistically driven approach moves away from a fixed number and requires a justification for the chosen sample size based on process knowledge and risk.

Principles of the Lifecycle Approach

This strategy is integral to the FDA's process validation lifecycle guidance, which comprises three stages [63]:

• Process Design: Building process understanding and identifying Critical Process Parameters (CPPs) that impact Critical Quality Attributes (CQAs).
• Process Qualification: Evaluating the process design to confirm it is capable of reproducible commercial manufacturing. The number of PPQ batches is determined here.
• Continued Process Verification: Ongoing monitoring to ensure the process remains in a state of control.

The number of batches required for Stage 2 (Process Qualification) is not predetermined. It depends on the level of process understanding gained during Stage 1 (Process Design). A process with less understanding or higher risk will require more statistical data (i.e., more batches) to confirm its consistent performance [64].

Statistical Justification Workflow

Determining the correct number of batches is a scientific exercise, as visualized in the following decision framework.

Diagram 2: Statistically Justified PPQ Batch Number Determination. This risk-based approach uses prior data and statistical goals to define the required number of validation batches (N).

Comparative Analysis: Consecutive vs. Statistical Approaches

The choice between these two strategies has significant implications for CGT development. The table below provides a direct, objective comparison.

Table 2: Objective Comparison of Validation Approaches

Aspect	Consecutive Batches (e.g., 3)	Statistically Justified Number
Regulatory Alignment	Aligns with general EU EMA expectations and traditional industry practice [2].	Aligns with US FDA's stated principles and the modern quality-by-design (QbD) paradigm [17] [63].
Statistical Rigor	Low. Based on a rule-of-thumb; may not adequately characterize process variability [65].	High. Sample size is chosen to achieve specific confidence levels and power, providing a data-driven assurance of consistency.
Flexibility for CGTs	Low. Inflexible for very small batch productions (e.g., ultra-rare diseases) or highly variable processes.	High. Can be adapted. May justify fewer batches for very consistent processes or more batches for highly variable ones.
Cost & Resource Impact	Predictable but potentially inefficient. Fixed cost for three batches, which may be insufficient or over-sufficient [64].	Variable but optimized. Aims to use the minimal number of batches needed for confidence, potentially saving resources long-term.
Handling of Validation Failure	Stringent. A single failure typically invalidates the entire series, requiring investigation and repetition [64].	Analytical. Failure is a data point that must be investigated and understood. The impact on the overall validation conclusion is assessed statistically.

Experimental Protocols for Validation Studies

The experimental workflow for generating validation data relies on robust, phase-appropriate analytical methods. For CGTs, this often requires orthogonal methods to fully characterize the product.

Protocol for Process Performance Qualification (PPQ)

• Objective: To demonstrate with a high degree of assurance that the commercial manufacturing process is reproducible and will consistently produce a drug product meeting all critical quality attributes.
• Methodology:
  • Protocol Development: Create a detailed PPQ protocol defining the manufacturing process, operational parameters, in-process controls, acceptance criteria for CQAs, and the statistical plan for data analysis.
  • Batch Execution: Execute the predefined number of PPQ batches (N) under normal commercial production conditions, using trained personnel, standard operating procedures, and commercial-grade equipment and facilities.
  • Data Collection & Analysis: Perform extensive testing on the PPQ batches. For a statistically justified approach, use statistical tools (e.g., process capability analysis - Cpk, confidence intervals) to evaluate if the process is in a state of control relative to the pre-defined acceptance criteria.
  • Report: Generate a final report that concludes whether the process has been successfully validated.

Protocol for Analytical Method Validation

Robust analytics are non-negotiable for batch validation. A common CMC deficiency for CGTs is an unvalidated potency assay [4].

• Objective: To ensure that analytical methods used to test PPQ batches are suitable for their intended purpose and provide reliable data.
• Methodology: The methodology should follow ICH Q2(R2) guidelines, with phase-appropriate implementation [1].
  • For early-phase (IND), methods need to be qualified (reliable and reproducible).
  • For late-phase (BLA/MAA), full validation is required, assessing parameters like:
    • Accuracy: Closeness to a true value.
    • Precision: Repeatability and intermediate precision.
    • Specificity: Ability to measure the analyte in the presence of impurities.
    • Linearity & Range: The interval where method performance is linear.
    • Robustness: Reliability under small, deliberate variations.

For CGTs, the FDA and EMA encourage the use of orthogonal methods (methods based on different scientific principles) to measure the same CQA. For example, vector copy number might be assessed using both qPCR and next-generation sequencing (NGS) for greater confidence [1].

The Scientist's Toolkit: Essential Research Reagents & Solutions

Success in process validation depends on high-quality starting materials and robust analytical tools. The following table details key solutions for CGT process development and validation.

Table 3: Essential Research Reagent Solutions for CGT Validation

Reagent/Solution	Function in Validation	Critical Considerations
GMP-Grade Viral Vectors	Acts as a drug substance (FDA) or starting material (EMA) for genetically modified cells. Used in transduction.	Purity, potency, titer, and absence of replication-competent virus (RCV). Functional potency assays are critical for FDA [2] [1].
Characterized Cell Banks	The foundational biological source material (autologous or allogeneic).	Donor screening, identity, viability, and purity. Regulatory requirements for donor testing differ between the US and EU [2].
Cell Culture Media & Supplements	Supports the growth and maintenance of cells during the manufacturing process.	Formulation consistency, raw material sourcing, and absence of adventitious agents. Justification for all components is required in the CMC section [17].
Critical Analytical Assays	Measures CQAs for batch release and stability.	Potency Assays: Must be biologically relevant and functional [2] [4]. Identity/Purity Assays: Flow cytometry, PCR. Orthogonal Methods: Used to confirm key results [1].
Reference Standards & Panels	Serves as a benchmark for qualifying/validating analytical methods and for comparing batch-to-batch results.	Well-characterized and stable. Essential for demonstrating assay performance and product comparability [17].

The paradigm for batch validation in cell and gene therapy is shifting from a fixed, traditional rule toward a flexible, science-driven standard. While the "three consecutive batches" approach remains a defensible and commonly accepted baseline, particularly for EU submissions, the "statistically justified number" approach represents the modern regulatory standard advocated by the FDA and aligned with enhanced process understanding.

For CGT developers, the optimal strategy is not to choose one over the other absolutely, but to leverage the strengths of both within a robust, risk-based framework. Proactive engagement with regulators through pre-IND meetings (FDA) or scientific advice (EMA) is highly recommended to align on the chosen validation strategy early. By building a deep process understanding during development and justifying all CMC decisions with data, sponsors can navigate the nuanced US and EU regulatory requirements and accelerate the delivery of these transformative therapies to patients.

The regulatory landscape for cell and gene therapies, known as Cell and Gene Therapies (CGTs) in the United States and Advanced Therapy Medicinal Products (ATMPs) in the European Union, is characterized by both growing alignment and fundamental differences. This guide provides a comparative analysis of the Chemistry, Manufacturing, and Controls (CMC) requirements from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The analysis reveals significant convergence in overarching principles and risk-based approaches, yet persistent divergence in technical areas such as the classification of starting materials, donor testing, and specific potency assay expectations. Understanding these nuances is critical for efficient global development strategies.

The development of cell and gene therapies is a global endeavor, yet sponsors must navigate two distinct regulatory paradigms [11]. In the United States, the FDA's Center for Biologics Evaluation and Research (CBER) regulates these products under the umbrella term Cell and Gene Therapies (CGTs) [19] [11]. The primary regulatory guidance for their development is provided through a growing suite of product-specific and general guidances, many of which have been recently updated or drafted, such as the 2023 draft guidance on "Manufacturing Changes and Comparability for Human Cellular and Gene Therapy Products" [19].

In the European Union, the EMA governs these innovative treatments under the regulatory framework for Advanced Therapy Medicinal Products (ATMPs), which are formally categorized into gene therapy medicinal products (GTMPs), somatic cell therapy medicinal products (sCTMPs), tissue-engineered products (TEPs), and combined ATMPs [5] [1]. A significant recent development is the adoption of the new "Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials," which came into effect in July 2025 [14]. This multidisciplinary guideline consolidates information from over 40 previous documents and serves as a primary reference for clinical trial applications [14].

Comparative Analysis of Key CMC Areas

The following sections and tables provide a detailed, side-by-side comparison of the FDA and EMA positions on critical CMC topics, highlighting both convergent and divergent expectations.

CMC Consideration	US FDA Position	EU EMA Position
Starting Materials	No formal regulatory definition; uses "critical raw materials" [2]. Expects an enhanced material control approach aligned with risk and development stage [2].	Precise definition; materials that become part of the drug substance (e.g., vectors) must be produced under GMP principles [2] [1].
Viral Vector Classification	Classified as a drug substance [2] [11].	Often classified as a starting material, particularly for ex vivo genetically modified cells [2] [11].
Potency Testing for Viral Vectors (in vitro)	Requires a validated functional potency assay for pivotal studies [2].	Infectivity and transgene expression may be sufficient in early phases; functional assays expected later [2].
Donor Testing	Governed by 21 CFR 1271. Must be tested in CLIA-accredited labs [2].	Governed by the EUTCD and new SoHO (Substances of Human Origin) legislation. Must be handled in licensed premises [2] [1].
GMP Compliance	Phase-appropriate approach, moving from "fit-for-purpose" to full GMP compliance verified via pre-license inspection [11] [14].	GMP compliance is mandatory for clinical trial materials, verified through self-inspections and regulatory oversight [14].
Process Validation (PV) Batches	Number not specified; must be statistically adequate [2].	Generally, three consecutive batches are required, with some flexibility [2].
Use of Platform Data in PV	Acceptable where similar manufacturing steps are used [2].	Acceptable where similar manufacturing steps are used [2].
Stability Data for Comparability	Thorough assessment including real-time data for certain changes [2].	Real-time data not always needed; accelerated studies can be sufficient [2].
Regulatory Interaction	INTERACT, Pre-IND, Type B, C, D meetings available [1] [11].	National and EMA-level scientific advice, CAT classification procedure [1].

Alignment and Convergence

A clear trend of regulatory convergence is evident in several strategic areas. Both agencies emphasize a risk-based approach to evaluating the impact of manufacturing changes on quality attributes [2]. There is also a shared understanding that the extent of analytical testing for comparability should increase with the stage of clinical development [2]. Furthermore, both regulators acknowledge the utility of accelerated or stress stability studies for identifying differences in stability-indicating attributes and accept the use of platform data for process validation when the same or similar manufacturing steps are employed [2]. The adoption of orthogonal analytical methods (methods using different scientific principles to measure the same attribute) is encouraged by both the FDA and EMA to build confidence in critical quality attributes, especially where standardized assays are lacking [1].

Persistent Divergence

Despite these areas of alignment, significant differences remain that can complicate global CMC strategy.

• Starting Materials and Viral Vectors: This is one of the most impactful areas of divergence. The EMA has a formal definition for 'starting materials' (materials that are intended to become part of the drug substance) and requires them to be manufactured in accordance with GMP [2]. Consequently, viral vectors for ex vivo modification are often considered a starting material in the EU. In contrast, the FDA classifies these same viral vectors as a drug substance, subject to more stringent oversight throughout development [2] [11].
• Potency Assay Requirements: While both agencies stress the importance of potency testing, their expectations for viral vectors used in vitro differ. The FDA expects a validated functional potency assay to be essential for assessing the efficacy of the drug product used in pivotal studies. The EMA, however, may find infectivity and expression of the transgene sufficient in early phases, with less emphasis on highly functional assays in later stages [2].
• Donor Eligibility and Testing: Both regions have rigorous donor testing requirements, but they are governed by different legal frameworks and technical expectations. The FDA's requirements are detailed in 21 CFR 1271, with testing expected to be performed in CLIA-accredited laboratories. The EU's requirements are based on the European Union Tissues and Cells Directive (EUTCD) and the new SoHO legislation, which mandates that activities are handled in licensed and accredited centers [2] [1]. The EMA also requires some donor testing for autologous material, which is a key difference from the FDA [2].
• GMP Compliance Timelines: The EU requires GMP compliance for manufacturing investigational medicinal products used in clinical trials [14]. The FDA, however, applies a more phase-appropriate approach, where full GMP compliance is verified later via a pre-license inspection, while early phases must be "fit-for-purpose" with an emphasis on patient safety and sterility [11] [14].

Experimental Protocols and Data Analysis

Protocol: Designing a Comparability Study Following a Manufacturing Change

Objective: To demonstrate that a manufacturing change does not adversely impact the critical quality attributes (CQAs), safety, or efficacy of the cell therapy product.

Methodology:

• Risk Assessment: Identify all CQAs potentially impacted by the change (e.g., identity, purity, potency, viability, vector copy number) based on prior knowledge and the nature of the change [2].
• Study Design:
  • Manufacture a minimum of three post-change batches [2].
  • Select an appropriate number of pre-change batches as a reference. Both agencies agree that the extent of testing increases with the clinical stage [2].
  • For the EU, if the change involves a recombinant starting material (e.g., a viral vector), the EMA guideline for genetically modified cells outlines specific attributes for both the starting material (e.g., full vector sequencing, absence of RCV, impurities) and the finished product (e.g., transduction efficiency, vector copy number) [2]. There is no direct FDA equivalent to this specific guidance.
• Analytical Testing:
  • Orthogonal Methods: Employ at least two complementary methods to measure key attributes like potency and identity. For example, use both qPCR and next-generation sequencing (NGS) for vector genome integrity [1].
  • Stability Studies: Include accelerated or stress stability testing to identify differences in stability-indicating attributes. The FDA may require real-time data for certain changes, while the EMA may not [2].
  • Potency Assay: Utilize a quantitative, biologically relevant functional assay that represents the product's mechanism of action. This is a common CMC deficiency noted by the FDA [66].
• Data Analysis:
  • Use statistical analysis to establish equivalence between pre- and post-change products.
  • For the EU, comparison to historical data is not required, whereas the FDA recommends its inclusion [2].

Data Presentation: Comparability Study Results

The following table summarizes hypothetical data from a comparability study for a hypothetical CAR-T cell product following a change in the cell culture process.

Table 2: Example Comparability Study Data for a CAR-T Cell Product

Quality Attribute	Test Method	Pre-Change Batches (n=3) Mean ± SD	Post-Change Batches (n=3) Mean ± SD	Acceptance Criterion Met?
Identity (% CD3+ cells)	Flow Cytometry	95.5% ± 2.1%	96.8% ± 1.5%	Yes
Potency (Cytokine Release, pg/mL)	ELISA (Functional)	1250 ± 150	1180 ± 130	Yes
Viability (%)	Trypan Blue Exclusion	98.2% ± 0.8%	97.5% ± 1.2%	Yes (≥70%) [66]
Vector Copy Number	qPCR (ddPCR orthogonal)	2.5 ± 0.3	2.6 ± 0.2	Yes
Purity (Endotoxin, EU/mL)	LAL Assay	<0.5	<0.5	Yes
Transduction Efficiency (%)	Flow Cytometry	75.4% ± 4.2%	72.1% ± 5.1%	Yes

Regulatory Pathway and Strategy Visualization

The following diagram illustrates the key regulatory interaction points and strategic considerations for navigating the US and EU CMC pathways, highlighting areas of convergence and divergence.

Diagram: CMC Regulatory Pathway for US and EU. This flowchart contrasts the phase-appropriate US FDA pathway with the GMP-first EU EMA pathway for CGTs/ATMPs, while highlighting strategic areas of convergence.

The Scientist's Toolkit: Essential Research Reagents and Materials

The complex development and testing of cell and gene therapies rely on a suite of critical reagents and materials, each with specific regulatory considerations.

Table 3: Key Research Reagent Solutions and Regulatory Considerations

Item	Function in CGT/ATMP Development	Key Regulatory Considerations
Viral Vectors (e.g., AAV, Lentivirus)	Delivery vehicle for gene transfer.	US: Drug Substance. EU: Starting Material. Requires full characterization (titer, identity, purity, potency) and testing for RCV [2] [66].
Plasmids	Vector construction and gene editing.	Used in generating GMP cell banks; history, derivation, and full sequencing are required [66].
Cell Banks (MCB/WCB)	Ensure a consistent and characterized cell source for manufacturing.	Full characterization is required, including identity, viability, purity (sterility, mycoplasma), and tumorigenicity [66].
Cell Culture Media & Reagents	Support cell growth, expansion, and differentiation.	All reagents (e.g., cytokines, growth factors, serum) must be qualified, and methods to demonstrate clearance of reagents from the final product are expected [66].
Gene Editing Machinery (e.g., CRISPR-Cas9)	Enables precise genomic modifications.	EU: Defined as a starting material, requiring GMP-grade manufacturing for investigational products [1]. Functional characterization and off-target analysis are critical.
Flow Cytometry Antibodies	Critical for identity testing (e.g., immunophenotyping) and potency assays.	Assays must be qualified (early phase) and validated (late phase). Antibodies used should be well-characterized for specificity and reproducibility [66].
qPCR/ddPCR Reagents	Used for vector copy number analysis, residual DNA testing, and mycoplasma detection.	Methods should be orthogonal where possible (e.g., qPCR and NGS). Assay validation includes accuracy, precision, and specificity [1].

The regulatory landscape for cell and gene therapies is dynamic, with a clear trend toward strategic convergence in risk-based approaches and data requirements, yet persistent technical divergence in areas like starting materials and GMP timing. The recent EMA clinical trial guideline and the ongoing development of an ICH Q5E Comparability Annex for ATMPs signal a move toward greater harmonization [2] [14]. For researchers and developers, success in this environment requires a nuanced, region-specific CMC strategy that leverages areas of alignment while proactively managing points of divergence through early and frequent dialogue with both the FDA and EMA.

Conclusion

Successfully navigating the EU and US CMC landscape for cell therapies demands a nuanced, strategic approach that recognizes both convergence and divergence. Key takeaways include the critical need to define starting materials correctly for each region, develop robust, functionally relevant potency assays, and implement a risk-based control strategy from the outset. While the EMA's new multidisciplinary guideline and FDA's evolving draft guidances show promising regulatory alignment, significant differences remain in GMP application, donor testing, and specific testing requirements. Future success will hinge on developers' abilities to design global programs with regional flexibility, engage regulators early and often, and invest in scalable, well-controlled manufacturing processes. As the field advances, embracing platform data, alternative analytical methods, and proactive comparability protocols will be essential for accelerating the delivery of these transformative therapies to patients worldwide.

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