Rigorous quality control is essential for any LNP-based experiment: batch-to-batch variability in particle size, surface charge, RNA encapsulation, and — most critically — biological activity can profoundly affect experimental outcomes and complicate interpretation of cell culture or animal data. Our QC workflow goes beyond standard physicochemical characterization by taking into account a specific activity that directly links the biological potency of an LNP batch to its particle concentration, thus enabling true quantitative comparability across different formulations and time points.
All EUFECT® LNP batches are characterised by Nanoparticle Tracking Analysis (NTA) using the ZetaView® system (Particle Metrix). By tracking individual particle Brownian motion under laser illumination, NTA simultaneously delivers three critical parameters from a single measurement run:
Confirms successful nanoparticle assembly (target: 80–160 nm) and flags aggregation or polydispersity.
Confirms adequate PEG-lipid surface coverage and colloidal stability (target: -1 to -20 mV at physiological pH).
Absolute particle count (particles/mL) — the critical denominator in the specific activity calculation.
Transfection efficiency is assessed by delivering eGFP reporter mRNA encapsulated in the LNP of interest into a defined cell type under standardised conditions. After 18–24 hours, cells are analysed by flow cytometry. The Mode of the fluorescence intensity distribution — the most frequently occurring fluorescence intensity value across all measured cells — is used as the primary expression readout, as it robustly represents the central tendency of the eGFP signal in the cell population. This approach provides a robust measure of the characteristic expression level of transfected cells while minimizing the influence of non-transfected cells and outliers. In addition, the percentage of eGFP-positive cells is reported to quantify the overall transfection efficiency.
The Mode represents the peak of the fluorescence intensity distribution and is insensitive to rare outlier cells or debris, making it more robust than the Mean for batch-to-batch comparisons.
The specific activity enables direct, quantitative ranking of LNP batches that would otherwise be difficult to compare: two formulations may show similar Mode values while differing three-fold in particle concentration, meaning one achieves the same biological output with far fewer particles — a critical distinction when optimising formulation efficiency or planning in vivo dose escalation. Conversely, a batch with high particle count but a low Mode signals poor RNA loading, incomplete endosomal escape, or RNA degradation during preparation.
ZetaView® NTA provides size distribution, zeta potential, and absolute particle concentration — all from a single measurement, enabling normalisation of biological data to particle number rather than RNA mass.
Specific activity (Mode / particle concentration) provides a single, potency value per batch enabling robust batch release decisions and longitudinal stability assessment, while minimizing confounding effects arising from cell-type-specific differences in uptake and expression.
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