RP-CAD for Lipid Quantification: Systematic Method Development and Intensified LNP Process Characterization

Area of Science:

• Analytical Chemistry
• Biopharmaceutical Manufacturing
• Nanotechnology

Background:

• Lipid nanoparticles (LNPs) are crucial for nucleic acid delivery, but their quality assessment often focuses on particle size and encapsulation efficiency.
• A need exists for comprehensive methods to characterize LNP composition and monitor manufacturing processes.
• Current LNP characterization methods may not fully capture lipid content and process-related variations.

Purpose of the Study:

• To develop and validate a holistic reversed-phase (RP)-charged aerosol detection (CAD) method for LNP and process characterization.
• To apply the RP-CAD method to evaluate process parameters, specifically total flow rate (TFR), during microfluidic mixing.
• To identify sources of lipid deviation and loss during LNP manufacturing.

Main Methods:

• Development of an RP-CAD method with optimized power function value (PFV) using exploratory calibration.
• Method validation including linearity (R² > 0.996), precision, accuracy, and robustness testing.
• Application of the RP-CAD method to analyze LNP processing under varying TFR during microfluidic mixing.

Main Results:

• The RP-CAD method demonstrated excellent linearity, precision, accuracy, and robustness for quantifying six common LNP lipids.
• Analysis revealed a constant lipid molar ratio during microfluidic mixing, independent of TFR.
• Deviations in lipid content were traced to lipid stock solution preparation, and lipid loss was attributed to post-mixing dialysis, both TFR-independent.

Conclusions:

• The developed RP-CAD method provides a robust tool for lipid quantification throughout LNP processing.
• This method enables detailed LNP characterization and process performance evaluation.
• The findings highlight the potential for RP-CAD in optimizing LNP manufacturing and quality control, applicable to various LNP formulations and processes.