Rational design and robust formulation processes are critical for optimal mRNA delivery by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can impact their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of sub-populations of mRNA-LNPs. Four LNP formulations with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of “fractions” was observed across the gradient, from the less dense top-most fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses.
Parent LNPs, A-DOPE and B-DOPE, resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the top-most fraction compared to the lower one, indicating some degree of heterogeneity; while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their sub-populations.
