Table 9.

Challenges of manufacturing of MSC-EV- based products for clinical application

Manufacturing Process Development of MSC-EV based Product
A. Justification of specifications and release criteria
Criterion	Description	Methods used for EVs (Pharpacopoeial texst if available)	Research status on EVs	Acceptance criteria
Identity	Confirmation that the preparation contains the correct cell-derived EVs with characteristic markers and parameters.	Western blot / ELISA (markers CD9, CD63, CD81, TSG101, Alix); NTA (size, particle number); TEM / Cryo-EM (morphology); Bead-based flow cytometry (ExoView, MACSPlex).	Well-developed studies; adherence to MISEV2023 standards.	Acceptance criteria limits/ranges necessary to determine.
Purity	Assessment of the proportion of actual EVs relative to contaminants (proteins, lipids, microparticles, culture medium residues).	NTA (particle/protein ratio – purity index); Western blot for negative markers (calnexin, GRP94); SEC chromatography and protein profile analysis.	Lack of full standardization.	Acceptance criteria limits/ranges necessary to determine.
Potency	Confirmation of biological activity consistent with the drug’s mechanism of action (e.g., regeneration, immunomodulation).	In vitro functional assays (HUVEC migration, fibroblast proliferation, cytokine inhibition); Immunological tests (T cell suppression, NK cell activation); Measurement of effector gene or protein expression (RT-qPCR, ELISA).	Many studies are being conducted; however, there is no universal test, and each application requires its own.	Acceptance criteria limits/ranges necessary to determine.
Safety	Absence of bacteria, fungi, mycoplasma, and endotoxins in the product.	Sterility test (Ph. Eur. 2.6.1); LAL test for endotoxins (Ph. Eur. 2.6.14); PCR/mycoplasma culture (Ph. Eur. 2.6.7).	Test methods internationally harmonized outlined in pharmacopoeias like the USP and European Pharmacopoeia (Ph. Eur.), requiring validation for EVs based products.	Well defined acceptance criteria limits/ranges: the EV- based products has to be sterile, negative for mycoplasma and viruses; endotoxin limits depend on the administration routes (ex. 5EU per kg body weight per hour for intravenous administration).
	Absence of adventitious viruses in source cells, raw materials, and the product.	Viral safety (Ph.Eur 5.1.7)

B. Justification of the storage conditions and batch to batch consistency
Criterion	Description	Methods used for EVs	Research status on EVs
Stability	Determination of the stability of the EV- based product during storage and transport.	Stability studies at different storage temperatures (4 °C, -20 °C, − 80 °C) and time points. Analysis of freeze-thaw cycles. Confirmation of the acceptance criteria for identity, purity, potency and safety tests following EV-based product storage.	Limited data, single studies.
Batch- to bach consistency	Determination of the batch to bach consistency of EV-based product.	Confirmation of the acceptance criteria for identity, purity, potency, and safety tests for EV-based products obtained from different donors.	Limited data, single studies. The properties of MSC-EVs may differ depending on donor. There are no clear inclusion/exlusion criteria of MSC-EV donor.
C. Non-clinical studies
Criterion	Description	Methods used for EVs	Research status on EVs
Pharmacokinetics and biodistribution	Physical and chemical characterization of the particles.	Labelling of EVs with fluorochromes (DiR, PKH26); Isotope labelling (e.g., 99mTc, ¹¹¹In); Reporter protein labelling (luciferase, GFP).	Limited data, single studies. There are technical limitations in the evaluation of pharmacokinetics for EVs-based product. Artificially-labelled EVs may not reflect physiological dynamics by any visualization method.
Toxicology	Evaluation of acute and repeated- dose toxicity following single or multiplate EV based product administrations	In vivo single and repeated administration, single-dose studies with an appropriately extended post-dose observation; histopathological and biochemical analysis of major organs.	Limited data available. Only isolated short-term observations; comprehensive long-term safety studies are urgently required for regulatory approval.
Cancirogenity/tumourigenicity	Evaluation of the potential of EV- based product to promote uncontrolled cell proliferation or tumor formation.	Long-term animal studies assessing tumor incidence and histopathology; expression of proliferation markers (Ki-67, PCNA); oncogene and tumor suppressor gene expression profiling.	No systematic data available; carcinogenic risk remains unexplored. Long-term safety testing is required prior to clinical translation.
Genotoxicology	Assessment of EV capacity to induce DNA damage or chromosomal instability.	In vitro comet assay, micronucleus test; in vivo genotoxicity models assessing chromosomal aberrations.	No validated data for EVs; genotoxicity testing not yet performed in standardized systems.
Immunotoxicity and immunogenicity	Assessment of immune system activation, cytokine response, and potential immunotoxic effects following EVs administration.	Cytokine release assay; immune cell activation Histological analysis of immune system activation both locally and systemically.	Preliminary studies indicate low immunogenicity, an inhibitory effect on inflammation induced by various factors, but lack of systematic evaluation under GMP conditions.