Analytical approach for identification and mechanistic insights into mRNA-lipid adduct formation

Messenger ribonucleic acid (mRNA), a promising tool in vaccine and therapeutic development, is reliant on intact mRNA delivery into target cells. Given its susceptibility to degradation, ensuring its stability is crucial, necessitating rigorous quality control throughout the product life cycle. This study presents an ion-pair reverse-phase liquid chromatography method that enables rapid and direct mRNA extraction from lipid nanoparticles, facilitated by using a surfactant in the sample preparation. This method, optimized using design of experiments (DoE), allows relative quantification of intact mRNA, mRNA fragments, and mRNA-lipid adducts. Forced degradation studies were used to investigate the impact of mRNA-lipid adducts on protein expression and to identify their chemical structures. The structures, identified by mass spectrometry, suggest reaction mechanisms that differ from those described in the literature so far. Further studies evaluated how formulation parameters such as pH, ionic strength, and buffering species affect mRNA-lipid adduct formation and mRNA fragmentation. A DoE assessed the impact of formulation parameters on mRNA integrity and mRNA-lipid adducts, showing that pH plays the major role. Overall, these findings have significant implications for the design and development of future mRNA-based biopharmaceuticals.