Measuring ethanol clearance in lipid nanoparticles and liposomes by benchtop NMR.

A benchtop nuclear magnetic resonance (NMR) method for quantifying ethanol in liposomal emulsions and mRNA-lipid nanoparticle (mRNA-LNP) systems is presented. The method development performed demonstrates the feasibility of benchtop NMR as an alternative to gas chromatography (GC) for ethanol quantitation, offering advantages such as simpler operation, reduced instrument maintenance, and minimal sample preparation. The use of benchtop NMR was assessed in terms of accuracy, linearity, precision, repeatability, and sensitivity with the limit of quantitation (LOQ) being ≤ 25 mg/mL, which is well below the necessary 5000 mg/mL value established for Class 3 solvents by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). This study also highlights the utility of benchtop NMR as a process analytical technology (PAT) tool, providing insights into the ethanol clearance efficiency of the mRNA-LNP process from initial lipid dissolution to the end of the purification steps that lead to process modifications. The method shown here can be applied to measuring ethanol in various lipid formulations as well as many other matrices containing large molecules that often interfere in other methods.