Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations

Abstract

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10–25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

The current landscape of nucleic acid therapeutics

“…Rapid-mixing techniques produced LNP− siRNA systems with high entrapment efficiencies (>85%) and narrow size distributions. Recent work suggests that such structures include a hydrophobic oil core primarily consisting of neutral ionizable lipid surrounded by siRNA complexed to lipids in a bilayer arrangement 76 with heterogeneity in the siRNA copy number per particle 77 .…”

Particles in Biopharmaceutical Formulations, Part 2: An Update on Analytical Techniques and Applications for Therapeutic Proteins, Viruses, Vaccines and Cells

“…Like for other nanoparticles (cryo-)EM, AFM and DLS allow sizing of LNP in the ensemble of the formulation. In addition, techniques in which the particles are separated first and then analyzed, e.g., AF4 197 , SEC 198 , AUC 199 are applied. SV-AUC was used to measure differences in mRNA loading by applying density matching with D 2 O.…”

“…SV-AUC was used to measure differences in mRNA loading by applying density matching with D 2 O. 199 Apart from density variations, particle size and molar mass could also be determined with SV-AUC.…”

Reincarnation of the Analytical Ultracentrifuge: Emerging Opportunities for Nanomedicine

“…In another more detailed study, lipid NPs were investigated regarding their partial specific volume, υ, that may change significantly once serving as a carrier for, e.g., genetic material. 70 The authors approached this challenge by solvent density matching experiments. In this case, different ratios of buffered water and heavy water have been used, and the sedimentation behavior was investigated.…”