Table 6 Overview of holistic quality control strategies for EV drugs
Category | Key Elements | Key Points |
|---|---|---|
Production materials | Cells for production | Refer to Cellular & Gene Therapy Guidances and ICH Q5D |
Raw materials (natural EVs) | Cells, culture media, additives | |
Raw materials (engineered EVs) | Plasmids, bacteria, auxiliary cells, viruses, small-molecule drugs, bioactive molecules | |
Raw materials, excipients, consumables, packaging materials | Traceable, controllable, and sourced; nonanimal origins recommended; refer to Cellular & Gene Therapy Guidances | |
Critical process parameters and in-process controls | Key steps in production, IPCs, intermediate testing (if applicable) | Define CPPs and IPCs using quality by design principles to ensure EV consistency and quality; include process performance indexes, microbial safety testing, and other appropriate IPCs |
Characterization of EV drugs | Identity | Surface molecular markers (e.g., CD9, CD63, CD81, ALIX), STRs, target molecule identification (for engineered EVs) |
Particle size and concentration | Size and quantity of EV particles | |
Morphology and structure | Primary assessment of integrity and purity | |
Zeta potential | Surface charge analysis | |
Component analysis | Lipids, proteins, nucleic acids (including omics analysis); difficult to exclude interference from coisolates during EV isolation and purification | |
Key drug components | Define and detect functional molecules (e.g., miRNA, proteins) | |
Purity, contents | Purity may be determined based on membrane integrity and marker positivity; evaluate encapsulation efficiency, loading capacity, and positive rate (for engineered EVs) | |
Impurities | Nonmembrane particles, free molecules, nonpositive particles, and drug-free EVs based on product function; Free molecules, nonmembranous particles defined as impurities based on product function | |
Biological activity | Based on mechanism of action; evaluated at molecular, cellular, and animal levels; e.g., immunoregulatory, anti-apoptotic, pro-angiogenic (for natural EVs); uptake efficiency, therapeutic molecule function (for engineered EVs) | |
General physicochemical properties | pH, osmotic pressure, visible foreign matter, and microbial safety-related quality attributes | |
Specifications | General considerations | Establish release and stability specifications for drug substance and product (if applicable); test items should generally include identity, particle size distribution and concentration, morphology, biological activity, purity and impurities, microbiological safety attributes, and general attributes; analysis methods should be fully validated |
Acceptable criteria | Determined based on nonclinical and clinical studies; batch release and stability study data combined with statistical methods | |
Engineered EV-specific attributes | Encapsulation efficiency, drug load, positive rate | |
Reference standards | Encourage reference standards development | |
Stability studies | Storage, packaging materials, transport | Support shelf-life determination; evaluate stability of intermediates |
Process validation | Batch consistency; quality controllability | Validate with ≥3 consecutive batches; usually not validated for virus clearance |
