Anion exchange-HPLC method for evaluating the encapsulation efficiency of mRNA-loaded lipid nanoparticles using analytical quality by design

Lipid nanoparticles (LNPs) are emerging nucleic acid delivery systems in the development of mRNA therapeutics such as the severe acute respiratory syndrome coronavirus 2 vaccines. However, a suitable analytical method for evaluating the encapsulation efficiency (EE) of the LNPs is required to ensure drug efficacy, as current analytical methods exhibit throughput issues and require long analysis times. Hence, we developed and validated an anion-exchange HPLC method using Analytical Quality by Design. Three critical method parameters (CMPs) were identified using risk assessment and Design of Experiments: column temperature, flow rate, and sodium perchlorate concentration. The CMPs were optimized using Face-Centered Central Composite Design. The discriminating power of the optimized HPLC method and RiboGreen assay was comparable. The main advantage of this method is that LNPs can be directly injected into the HPLC system without bursting the LNPs loaded with encapsulated poly(A). The optimized HPLC method was validated as robust, high-throughput, and sufficiently sensitive according to the ICH Q2 guidelines. We believe our findings could promote efficient LNPs-based drug development.