Advanced Biophysical Analysis Reveals Critical Differences in Leading Lipid Nanoparticle Formulations

Comparative Analysis of Clinically Significant Lipid Nanoparticles

Researchers have conducted an extensive biophysical characterization of four clinically important lipid nanoparticle (LNP) formulations, according to reports published in Nature Biotechnology. The study compared LNPs containing ionizable lipids MC3 (used in Alnylam’s Onpattro), C12-200 (academic standard), SM-102 (Moderna’s Spikevax), and ALC-0315 (Pfizer/BioNTech’s Comirnaty), all prepared with modified mRNA encoding firefly luciferase.

Comparative Analysis of Clinically Significant Lipid Nanoparticles
Manufacturing Methods Significantly Impact LNP Properties
Advanced Techniques Reveal Structural Variations
Internal Structure and Morphology Differences
Shape and Cargo-Dependent Structural Changes

Sources indicate that these formulations represent gold standards in therapeutic delivery, with MC3 being the first siRNA-LNP therapy approved for hereditary transthyretin-mediated amyloidosis, while SM-102 and ALC-0315 form the backbone of major COVID-19 vaccines. The C12-200 formulation serves as an academic benchmark for nucleic acid delivery., according to industry analysis

Manufacturing Methods Significantly Impact LNP Properties

The report states that formulation methodology dramatically affected LNP characteristics. Microfluidic-formulated LNPs demonstrated superior performance with mRNA concentrations of 40-60 ng/μl and encapsulation efficiencies exceeding 80%, while bulk-mixed LNPs showed lower concentrations of 10-30 ng/μl and approximately 50% encapsulation efficiency. Analysts suggest that C12-200 LNPs were an exception, maintaining consistent mRNA concentration across both manufacturing methods.

According to the analysis, microfluidic-formulated particles exhibited smaller hydrodynamic radii of approximately 40 nm with higher particle concentrations, compared to bulk-mixed LNPs which had larger radii around 100 nm and lower concentrations. All LNPs maintained neutral ζ-potential and apparent pKa values around 6, the report indicates.

Advanced Techniques Reveal Structural Variations

Researchers employed multiple sophisticated analytical techniques, including sedimentation velocity analytical ultracentrifugation (SV-AUC) and field-flow fractionation with multi-angle light scattering (FFF-MALS). The data revealed that MC3, SM-102, and ALC-0315 LNPs predominantly floated during centrifugation, while C12-200 LNPs sedimented, correlating with their respective lipid densities.

Analysts suggest that microfluidic-formulated samples displayed significantly less polydispersity than bulk-mixed samples across all characterization methods. The report states that FFF-MALS provided more accurate dispersity measurements than traditional dynamic light scattering, better resolving the true heterogeneity of LNP populations.

Internal Structure and Morphology Differences

Small-angle X-ray scattering (SAXS) combined with size exclusion chromatography and singular value decomposition revealed detailed internal structures. According to reports, all RNA-loaded LNPs exhibited characteristic Bragg peaks at scattering vectors between 0.10-0.16 Å^-1, corresponding to ordered internal structures with spacing of approximately 40-63 Ångströms.

The study found that C12-200 LNPs displayed distinct multilamellar ring structures not observed in other formulations, which typically contained amorphous cores with exterior bilayers. Sources indicate that this uniqueness stems from C12-200’s shorter alkyl tails and piperazine core structure, contrasting with other lipids featuring single nitrogen atoms and longer alkyl chains.

Shape and Cargo-Dependent Structural Changes

Researchers determined that microfluidic-formulated LNPs adopted prolate ellipsoidal morphologies, with shape factors ranging from approximately 2.9 for MC3 and SM-102 to 3.1 for C12-200 and ALC-0315. The report states that C12-200 exhibited the greatest anisotropy among the formulations tested.

When comparing different cargo types, analysts suggest that MC3 formulations containing siRNA demonstrated more intense Bragg peaks than mRNA-loaded versions, indicating that LNP internal structure is sensitive to both cargo type and manufacturing method. This finding suggests that optimal formulation parameters may vary depending on the specific therapeutic nucleic acid being delivered.

The comprehensive analysis, according to researchers, provides crucial insights for optimizing LNP design and manufacturing processes, potentially informing future development of more effective nucleic acid therapeutics and vaccines.