Assessing the Heterogeneity Level in Lipid Nanoparticles for siRNA Delivery: Size-Based Separation, Compositional Heterogeneity, and Impact on Bioperformance

Abstract

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60-190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60-150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.

Nanoparticle heterogeneity: an emerging structural parameter influencing particle fate in biological media?

“…The two NPs batches without fractionation displayed overlapping dose-response curve. In contrast, fractions of the two batches showed significant differences for their in vitro activity, highlighting the importance of heterogeneity assessments 29 .…”

“…Recent reports have highlighted higher-than-expected variations in nanocarrier chemical composition correlated to their size polydispersity with important consequences on the performance. The influence of size polydispersity on the chemical composition and active delivery properties of lipid NPs encapsulating siRNA have been recently investigated 29 . Two similar lipid NP batches with similar bulk properties (chemical composition, hydrodynamic diameter, surface charge, and encapsulation levels) were tested.…”

A Single Dose of EGLN1 siRNA Yields Increased Erythropoiesis in Nonhuman Primates

“…Chemically modified double-stranded RNAs were synthesized and encapsulated in lipid nanoparticles at Merck Research Laboratories using the stepwise mixing and diafiltration method as described elsewhere [30,31]. The cationic lipid was synthesized at Merck (West Point, PA).…”

Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles: delivery and bioactivity in pancreatic cancer cells

“…2A)212223. Emerging studies indicate that the more homogeneous the nanoparticles are, the better their performance3132. So, to obtain liposomes with a homogeneous diameter, the solution was extruded 10 times through a double polycarbonate filter with pore size of 50 nm using compressed N 2 (20–40 bar)212223.…”