Standard Methods for Quality Control in Cell-Based Medicinal Products and their Validation for Clinical Applications

Abstract

In recent years, cell-based medicinal products (CMPs) have emerged as novel therapeutics with specific potential to treat a wide range of diseases. These products should be produced in accordance with good manufacturing practices (GMPs) and follow specific guidelines to ensure their safety and meet standard quality control criteria. This manuscript reviews current standard methods for quality control and validation specific to CMPs intended for clinical applications. We summarize critical quality attributes, including safety assessments such as sterility, endotoxin, mycoplasma, viral testing, tumorigenicity, and genetic stability; quantitative parameters, including cell counts and dose determination; and quality characteristics encompassing cell viability, morphology, growth kinetics, and immunophenotyping. We also address purity evaluation, potency assays, and the importance of validating analytical methods to guarantee reproducible and reliable test results. Furthermore, this article discusses necessary considerations for donor screening, raw material sourcing, manufacturing environment monitoring, and stability testing to maintain product integrity throughout production and storage. Emphasis is placed on adherence to GMP and relevant regulatory guidelines as defined by international pharmacopeias and authorities, such as the Food and Drug Administration and the European Medicines Agency. Finally, we highlight challenges faced in standardizing quality control for CMPs and underscore the need for continued development of rapid and robust testing methods tailored to their unique characteristics. This comprehensive overview aims to support academic and industrial stakeholders in implementing effective quality control strategies for advanced cell-based therapies.

INTRODUCTION

Rapid progress in the fields of biology, biotechnology, and medicine in recent years has resulted in the development of highly revolutionary medicinal products, including those containing cells and their derivatives for newly investigated therapies, and has shown tremendous potential to improve treatment protocols of various diseases.[1] In today’s medical world, regenerative medicine (RM) means “the process of repairing, replacing, bioengineering, or regenerating human cells, tissues, or organs to restore or establish their normal function.”[2] Good manufacturing practice (GMP) is a quality management system used to ensure that manufactured pharmaceutical products comply with defined standards, thus ensuring that the products are safe and of high quality, as much as possible. GMP was initially used to produce small molecules, vaccines, and monoclonal antibodies, but today, the same principles as GMP with some modifications apply to advanced therapy medicinal products (ATMPs), including gene therapy, somatic cell therapy, and tissue engineering products.[3,4] Cell-based medicinal products (CBMPs), which may be considered a subset of ATMPs, have been categorized into different groups based on their origin (allogeneic vs. autologous), combination with noncellular components, and applied genetic modifications. The possible risks for these products are not only dependent on the origin of cells but also on the manufacturing process and the materials used, manipulations, state of viability and proliferation of cells, excipients and noncellular components of the final products, their stability and storage conditions, and mode of administration.[1]

Mesenchymal stromal cells (MSCs) represent the most common CBMPs due to their availability, easy cellular derivation, high safety profiles, and lack of ethical issues.[5] The characterization of cell-based products, including specialized cells such as hepatocytes, requires standardized protocols to ensure reproducible identity, quality, and potency throughout manufacturing and clinical evaluation.[6] ATMPs, including gene and cell therapies, are increasingly being explored for neurodegenerative diseases such as Huntington’s disease, representing a promising translational approach for these complex disorders.[7] MSCs have been explored across a wide range of therapeutic areas, including regenerative dermatology, with advanced therapy medicinal products showing promise in conditions such as vitiligo.[8] MSC-based products with broad therapeutic applications have emerged as novel treatments in human healthcare in alignment with the different national or international regulatory authorities, such as the US Food and Drug Administration (FDA) and European Medicines Agency (EMA), with promising use in conditions ranging from dermatological disorders like vitiligo to liver diseases.[9] To release the qualified CBMPs as the most complex medical products, only authorized institutions are licensed to conduct relevant clinical trials and are primarily responsible for ensuring the quality of these products, which must abide by national regulations and guidelines.[10,11]

In addition to the regulatory environment, current quality control technologies are not effective enough to meet all quality control needs. For example, many aspects of the safety and biological efficacy of almost all CBMPs cannot be accurately assessed based on current technologies, and therefore, new evaluation techniques need to be developed.[11,12] In fact, the bioassays typically chosen to evaluate biological efficacy need to be validated by new preclinical and clinical data before their predictive functions can be developed for therapeutic efficacy. Moreover, rapid test methods are needed to evaluate the safety and efficacy of the products for the reasons listed. One of the most urgent needs is the establishment of rapid sterilization testing because the very short shelf life of the final products makes it impossible for conventional sterilization testing described in existing regulations/guidelines to meet the clinical needs of medicinal products derived from stem cells.[13,14]

Products derived from cells, tissues, or microorganisms, like their sources, have variable and intricate specifications, and special considerations are needed during manufacturing to ensure their consistency in product identity, quality, safety, purity, and biological properties. This step ensures that none of the produced products contain harmful substances or contaminants that could harm consumers. The parameters addressed in this process should be analyzed through validated methods encompassing every aspect of production.[15,16]

In our current manuscript, the sources and references were selected based on their relevance, scientific validity, and alignment with accepted guidelines and regulatory standards in the field of cell-based medicinal product quality control. The databases and sources, such as PubMed and regulatory agency publications, were searched using criteria such as publication type (review, original article, guidelines, and meta-analysis) and date range (2000–2025).

Quality control parameters

The quality control parameters chosen for a given cell substrate and the extent of characterization need to be decided according to the biological properties of the cells (e.g. growth pattern), its cultivation required material (including use of human-derived and animal-derived biological reagents),[17] and possible risks that may arise from in vivo use of the final product. Every characterization step should be performed by defined, validated analytical methods reviewed in different pharmacopeia or established in-house, and results should be compiled to evaluate their compliance with the relevant acceptance criteria. The most common guidelines are United States Pharmacopeia or USP, European Pharmacopoeia or Ph.Eur, International Pharmacopoeia or Ph.Int., and International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use or ICH, and can be taken as a reference based on the regulatory system of each country.[18] Despite the complexity of cellular products compared to other biological products, there are no globally approved standards and reference materials to facilitate their quality control and comparisons between different laboratories or institutions.[12] In addition, it is almost impossible to develop a single standard line to evaluate all qualitative aspects of their effectiveness, as they differ in their origin, stage, and differentiation potential, type of specific surface markers, and immunoregulatory effect.[14,19]

To ensure the quality of cell-based products and the safety and pharmaceutical efficiency of these products, various methods, including quality testing (QT) along with cell substance release testing (CSRT) and final product release testing (FPRT), should be employed.[14]

It should be emphasized that for autologous CBMPs without complex processing, the relatively less stringent specifications, maybe with one manual assay for each QT approach, are appropriate. However, for allogeneic CBMPs or autologous ones with complex processing, and for the banked cells, intense testing with more stringent specifications should be tested by at least two mutually supportive methods, and even product-specific assays should be employed. QT must be re-performed whenever changes occur with potential effects on product quality, such as significant changes in the production process or even the construction site and/or storage facility.[14,20]

As shown in Table 1, for appraising qualities of cell-based products, the following specifications should be addressed:

Table 1.

Quality control parameters. Each parameter consists of a test that can be assayed by one or several methods. The resulting data should be found within the acceptance criteria

Parameter	Test Name	Method/Assay	Acceptance Criteria
Safety	Sterility	Culture	No Growth
	Mycoplasma	Culture/PCR	No Growth/Not Detect
	Endotoxin	LAL (Gel Clot)	Product Specific
	Virus	PCR	Negative
	Tumorgenicity	In-vivo	Negative
	Genetic Stability	Karyotype/CGH Array	Normal
	TSE/BSE	Immunoassay	Negative
Quantity (Dose)	Cell Number	Product Specific	Product Specific
	Total DNA
	Total Protein
	Size
Quality	Cell Viability	Trypan Blue Exclusion/Cell Counter	≥70%
	Morphology	Microscopy	Product Specific
	Doubling Time	Cell Culture	Product Specific
	Senescence	Activity Assay	Acceptable
Identity	Morphology	Microscopy	Product Specific
	Immunophenotyping	Flowcytometry/IF	Product Specific
	HLA Typing	PCR	HLA Typing Should be Obtained
	DNA Profiling (STR)	PCR	Unique ID Should be Obtained
	Gene Expression at mRNA	RT-qPCR	Product Specific
	Protein Expression Quantification	Western Blot/Flowcytometry	Product Specific
Purity/Impurity	Detection of Undesired Cells, Animal or Human Cells Feeder Layers, …	Flowcytometry/PCR	Product Specific
	Detection of Unwanted Material (Animal Origin, Purified Enzymes, …)	ELISA/HPLC/ SDS PAGE
Potency/Efficacy	Biological Activity (Colony formation, Detection of Cytokine, Collagen, …)	Product Specific	Product Specific
Others	Appearance	Product Specific	Product Specific
	pH

1. Safety: Based on guidelines, safety assay is an important concern toward obtaining a uniform final product and freedom from adventitious agents.[21,22] The testing for adventitious agents should include tests for bacteria, fungi, mycoplasma, and viruses.

   • 1.1.Sterility assay: Microbial contamination of cellular products leads to inefficiency and adverse clinical events. Samples have to be inoculated in relevant culture conditions to survey for probable contamination in medicinal products. Fast and accurate detection of even low counts of both bacteria and fungi is necessary for rapid and proper action. As a result, the use of the BACTEC/ALERT system, which can detect aerobic and anaerobic bacteria, as well as fungal contamination, has been recommended. In line with raw material and product samplings, environmental monitoring also plays an important role in preserving the sterile condition during the manufacturing procedure.[23]

   • 1.2.Endotoxin test: The presence of endotoxin as the most potent pyrogenic contaminants in the manufacturing of products, a component of gram-negative bacteria, poses a great health risk when they come in contact with the bloodstream or cerebrospinal fluid,[10] and the lack of bacterial growth does not mitigate the probability of endotoxins in biological products.[24] Based on pharmacopeia guidelines, limulus amebocyte lysate testing, or LAL, uses an in vitro assay to detect and quantify endotoxin levels.[25] The level of medicinal products depends on the route of administration, the dose of the product administered per kilogram of body weight, and the duration (time) of administration.[24]

   • 1.3.Mycoplasma test: The mycoplasma genus is the smallest self-replicating prokaryotic organism and varies in size and morphology. Mycoplasma may cause serious contamination in cell and/or tissue cultures. So, detecting the mycoplasma is a necessary quality control requirement to ensure reliably pure and safe biotechnological products and raw materials. Cultured method and nucleic acid amplification technique (NAT), as a validated test, are common methods to detect mycoplasma contamination.[26]

   • 1.4.Viral safety test: Donors, cellular products, and raw materials with biological (human or animal) origin should be tested for a broad spectrum of relevant viruses by using appropriate screening tests before utilization. Documentation regarding the viral safety of materials with biological sources, which come into contact with medicinal products, should also be provided. Samples from donors and cell banks have to be screened for the presence of infectious materials and viruses: Human T-lymphotropic virus 1 (HTLV-1), human immunodeficiency virus 1 and 2 (HIV-1 and 2), Hepatitis A virus (HAV), Hepatitis C virus (HCV), Hepatitis B virus (HBV), human papillomavirus (HPV), Parvovirus B19 (PVB19), Epstein-Barr virus (EBV), cytomegalovirus (CMV), Rubella and Treponema Pallidum (syphilis), by immunoassay or molecular PCR to minimize the risk of viral transmission.[27,28,29,30]

   • 1.5.BSE/TSE: To avoid prions consumption, actions have to be taken to minimize the risk of transmission of animal spongiform encephalopathy. Materials with biological sources, e.g. fetal bovine serum or bovine collagen, should be purchased from countries where the presence of prions and bovine spongiform encephalopathy (BSE), belonging to the family of transmissible spongiform encephalopathies (TSEs), is routinely checked and properly documented.[27,30,31]

   • 1.6.Genetic stability evaluation: Different cells, such as mesenchymal stromal cells or fibroblasts, show abundant propagation skills. While it may be useful during production, increasing the number of cells and manipulations during culture will heighten the risk of DNA damage and genomic instability. Karyotype and comparative genomic hybridization (CGH) array, among other tests, help find any discrepancy that may affect treatment efficacy and patient safety.[32]

   • 1.7.Tumorgenicity test: Cells with high proliferation capacity may show their ability to form tumors when used in conditions like a compromised immune system. Besides, manipulations such as irradiation, by causing genomic instability, may activate the tumorigenic potential of cells. As a result, products have to be analyzed (in vitro or in vivo), and documents indicating their lack of tumorigenic ability are provided. To evaluate the tumorigenicity of cells, the soft agar colony formation assay is considered as an in vitro test,[33] and male mice with severe combined immune deficiency (SCID) are injected subcutaneously with stem cells and monitored weekly to assess in vivo tumor formation. After 4 months, probable tumors and major organs are stained by the hematoxylin and eosin (H and E) method and assessed.[10,18]

2. Quantity: Quantity or dose of products should be determined based on an appropriate assay. Where applicable, methods such as cell counts, using visual methods (trypan blue exclusion) or automated cell counters,[34] size of tissues and DNA or protein content,[21] in line with the route of administration, determine the dose of the products. Based on the determined dose (minimum effective dose and the optimal amount of administration) and validated measurements, a reference range should be calculated, and further sampling has to verify the quantity or dose of medicinal products.[35]

3. Quality: Quality of CBMPs depends on a wide range of parameters. Cell count, morphology, viability of cells, senescence, doubling time (growth rate), and pH are commonly analyzed:

   • 3.1.Morphology: Morphology can be considered a leading indicator of cell fate and differentiation stages. Culture conditions and the quality of cellular products directly affect the morphology of the cells and may cause them to differ from accepted conditions. Besides, it can be used to predict the differentiation ability of cells toward other cells.[22,36,37]

   • 3.2.Cell viability: It is necessary to ensure the state of viability in cells intended for therapies.[38,39] Nonviable cells, based on their administration purpose, may lose their functionality during in vitro assays, compromise the intended use of products, and change the prescribed dose of the drug substance. Loss of viability should be an indicator of changes in the culture of cells; hence, the reason should be determined. In this context, manual methods using trypan blue or automated analysis of viability using flow cytometry (with dyes 7-AAD and PI) and digital imaging are suggested to determine the product condition.[22,34]

   • 3.3.Growth rate: Evaluation of proliferation capacity is one of the most important factors in defining the quality of cells when stem cells are employed, and based on the stage of their differentiation, they may show different capabilities. Assessment of the proliferation ability of cells and senescence condition, performed at the initial stages of the production process, provides a great understanding of the quality of the cells.[8,21] Other additional tests, such as apoptosis, growth curve, and cell cycle assays, may be performed as a supplementary additional experiment to figure out the growth ability of cells. A cancer cell line, such as murine melanoma B16F10, can be used as a positive cell control due to its stable and high growth rate.[10,19]

4. Identity: An important part of the quality control system for cellular and biological products is verifying their identity. The defined tests, phenotypic or genotypic, have to be specific to the cells and encompass their unique characteristics and properties:

   • 4.1.DNA fingerprinting: DNA fingerprinting or short tandem repeat (STR) analysis provides a molecular-based profile of DNA extracted from cells and specifically displays the identity of cells.[21]

   • 4.2.Human leukocyte antigen (HLA) typing: The HLA system consists of major histocompatibility complex (MHC) proteins located on the surface of cells and plays a role in the compatibility of transplanted cells or tissues. Immune incompatibility when allogeneic cells or tissue are used is a contributor to transplant rejection. For this reason, molecular analysis of products and determination of HLA combination in cells reduces this risk. Although the use of allogeneic cells like MSCs, fibroblasts, and certain cells from the epidermis evokes minimal immune reactions, recipients overcome immune barriers, even in HLA-mismatched, for the use of allogeneic sources.[21]

   • 4.3.Morphology evaluation: Although the morphology of cells may change as a result of their culture condition and not define their identity thoroughly, cells of different origins are usually presented differently; MSCs and fibroblasts are spindle-shaped, while young and proliferative keratinocytes are cobble-stone-like cells that are tightly connected to each other. Where available, proper reference controls based on preclinical/clinical phase data or other developmental studies should be used to compare the morphology of cells in conjugation with other tests to affirm the identity of products.[21]

   • 4.4.Immunophenotyping test: Identity of cellular products can be determined by using antibodies against specific proteins expressed inside or on the surface of cells. Most of the cells have a specific pattern of expression for positive and negative markers. While immunostaining of the cells and tissue culture sections visually provides images of labeled cells and shows the specific location of markers’ expression, flow cytometry quantifies the amount of each marker. Based on the acceptance criteria defined for each cell type and pattern of expression, cells are characterized. MSCs, as a common cell therapy product, express three positive markers: CD90, CD73, CD29, and CD105 more than 95%, and lack the expression (<2%) of four hematopoietic markers: CD34, CD45, CD14 or CD11b, CD19 or CD79, and HLA-DR.[21,40]

   • 4.5.Gene expression analysis: Other than morphological and phenotypic characterization of cell-derived products, analyzing gene expression patterns by molecular methods complements the effort to identify the cells completely. Reverse transcription polymerase chain reaction (RT-PCR), or reverse transcription quantitative polymerase chain reaction (RT-qPCR), allows the use of RNA as a template. RNA transcripts are detected or quantified by reverse transcribing them into cDNA first using reverse transcriptase. Then, PCR or qPCR is subsequently done.[41]

     Like phenotypic markers, gene expression depends on the source and identity of cells and the conditions they are being cultivated. Therefore, by taking advantage of this trait, proper characterization of the products can be executed.[42,43]

5. Purity and impurities: During the preparation of cellular products, where a specific cell type is required, the unwanted cells are considered impurities. Their amount should be defined based on acceptance criteria, and the composition of the final product components should be completely described. On the other hand, nonviable cells may be considered critical as the integrity and biological activity of the cells are impacted by the viability of the products, irrespective of cell type.[1]

   In addition to evaluating the purity of products, manufacturers should also assess possible impurities. Every undesirable substance in products, being product- or process-related, excipients like buffers or degradation products are considered impurities, and their level should be determined by justified methods.[44] Remained residue of culture media and its components, enzymes, and buffers based on the risks they may pose should be thoroughly evaluated, and methods to remove their risk have to be devised when they exceed their acceptable range.[21]

6. Potency or Efficacy: Potency of a drug product is defined as the ability to show therapeutic activity when it is administered in controlled trials or tested in the laboratory in vitro or in vivo, or both. These tests are used to measure product attributes associated with product quality and manufacturing controls in vitro or in vivo. Efficacy data from clinical investigations can prove biological activity and potency in a product, and methods such as flow cytometry and gene expression profiles.[1] However, relevant data from these studies may not be available before the release of the product, and thus not a practicable method to quantitatively test for potency.[21,45]

   MSCs, as the leading cellular products, are multipotent cells that can differentiate toward chondrocytes, osteocytes, and adipocytes in relevant culture conditions. They are also known to have immunoregulatory effects that can be identified by their repression of lymphocyte proliferation[46] and release of a plethora of cytokines. Assays like mixed lymphocyte reaction and cytokine assay (inflammatory cytokine panel, LEGENDplex™) work by analyzing the effect of cells on lymphocytes’ proliferation and release of specific cytokines and determining the cells’ potency in vitro.[47] While these tests define MSCs’ immunological effectiveness, the tube formation assay can present the function of these cells in constructing tube-like structures, which form nodes and branches in specific media.[48]

7. Other characterizations: Other than cellular products, the visual appearance of the products (based on their color, texture, integrity, and opacity) and the integrity of the container closure system and their stability during storage, degradable materials in the final products should be assessed and analyzed.[21,49]

EQUIPMENT QUALITY CONTROL AND VERIFICATION

QC and verification of equipment are essential components of ensuring the reliability and consistency of cell-based medicinal products during both manufacturing and testing phases. This process encompasses regulatory compliance verification, the implementation of automated process control, and the application of scale-specific monitoring strategies.[50,51] QC of equipment demands verification of critical quality parameters, such as environmental conditions (temperature, humidity, pressure, CO₂ levels), cellular and metabolic markers, microbial, chemical, and genetic contaminants, alongside engineering factors. This verification should align with regulatory standards such as EU GMP, current GMP (cGMP), FDA/EMA directives, and quality by design criteria. For example, one study demonstrated facility qualification with continuous environmental monitoring, while others applied real-time process analytical technology and computational fluid dynamics for equipment characterization.[52,53] Automation and integrated process control emerge as essential tools. Studies on automated cell culture systems and single-use bioreactors utilized methods from physical tracer tests to advanced sensor networks, statistical process control, and model-based similarity to guarantee consistent operation.[54,55] The scale of operation influences strategic approaches: small-scale academic settings typically focus on environmental monitoring and practical contamination controls, whereas larger industrial applications emphasize risk-based frameworks, advanced analytics, and standardized methods for equipment verification.[53,56,57] Qualification procedures typically involve installation qualification, operational qualification, and performance qualification, which collectively ensure that each device functions according to established specifications and regulatory standards.[58] Adherence to GMP guidelines necessitates thorough documentation of all equipment QC activities, including calibration schedules, maintenance records, and corrective actions.[50]

Analytical method validation

During quality control and in-process control of products, every analytical method used should be well characterized, validated, and documented to reach credible results. Based on the type of the method, parameters including linearity, accuracy, precision, selectivity/specificity, sensitivity, reproducibility, and system suitability should be taken into accord and evaluated. These data should demonstrate the proposed testing and acceptance criteria and guarantee the reproducible quality of the products at the time of release and during their determined shelf-life.[21,59]

Product stability

Based on the type of the medicinal products, an adequately controlled storage condition should be established based on information about temperature, stress conditions, humidity, light, and closure system, which may affect cellular products to allow proper maintenance of cells without changing their intended final characteristics.[1,49] Furthermore, these data provide insights into the stability of products during storage and help to determine their shelf-life.

Tests and assays used for stability assessment depend mostly on the methods by which cells were cultivated and the nature of cells.[17] Generally, when working with cell-based products, storage conditions should be optimized to ensure the maintenance of viability, integrity, purity, sterility, and function of the products. Identity should be verified by relevant genotypic and/or phenotypic markers, and if suitable biochemical/immunological indicators. The desired product’s productivity has to be analyzed after storage to approve the functionality of the cells. Available data gained during stability studies at designated time points before use of the product have to be documented and employed to determine their specific shelf-life.[1,17]

Microbial monitoring of product manufacturing environments

Microbiological monitoring of the environment where the medicinal products are being produced effectively controls the manufacturing environment and reduces the risk of contamination. Monitoring programs directly depend on risk assessment and can facilitate early detection of potential problems.[60]

Among the most commonly used methods for monitoring aseptic environments are controlling air quality by particle counter, surface air system sampler, Settle plates, gelatin filter sampler, and analyzing equipment, facilities, and personnel using surface sampling.[1]

Tracing and control of raw materials

Assessment of starting material and excipients is one of the main tasks of the quality control units in the pharmaceutical industry and is essential for any production process to be reliable and lead to reproducible and consistent outcomes. It must be ensured that the necessary tests are applied to the incoming goods and the newly arrived raw materials are released only after their quality is confirmed. According to the current good manufacturing practice, all raw materials that come into contact with pharmaceutical products must be controlled and checked for purity, safety, efficacy, and identity. Also, an exact process should be in place to ensure raw material traceability. The ingredients of the culture medium must meet certain specifications, which need to be checked according to each ingredient and depending on the level of risk. This main goal can also be achieved by sampling, an important process in which only a small part of a batch is taken and analyzed based on the guidelines.

Raw materials with animal or human origin must be checked for TSE/BSE and viral safety, in addition to other routine tests such as sterility, endotoxin, mycoplasma, etc.[61]

Quality control challenges in cell-based medicinal products

While remarkable achievements have been made in cell therapy products, this industry still faces many challenges. Despite the introduction of guidelines and methods for analyzing quality control of CBMPs, the lack of cell and tissue products reference standards can be considered a major hurdle during each assay and its validation. Besides, there is not enough guidance for a defined impurity profile during the purity analysis of cell-based products, stability testing program criteria, and time points for defining their shelf-life. These challenges have led manufacturers to develop in-house product-specific reference standards for product quality control. At the same time, regional differences in regulatory approaches make different interpretations of GMP requirements possible for manufacturers, which clarifies the authority’s need for more precise and coherent regulatory approaches.[15,16,62]

CONCLUSION

During the cell manufacturing process, it is essential that strict quality control measures are adhered to, as a living and dynamic product by itself, and can be easily damaged or contaminated. So quality control and evaluation of the production play a crucial role in ensuring delivery of high-quality, safe, and efficient products. This study presented an overview of the general principles of key critical parameters for product assessment based on current guidelines for an advanced, effective, and well-organized quality planning process.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.