The development of lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics presents unique pharmaceutical and regulatory challenges. In contrast to small molecule drugs that are highly pure and well-defined, LNP drug products can exhibit heterogeneity in size, composition, surface property, or morphology. The potential for batch heterogeneity introduces a complexity that must be confronted in order to successfully develop and ensure quality in LNP pharmaceuticals. Currently, there is a lack of scientific knowledge in the heterogeneity of LNPs as well as high-resolution techniques that permit this evaluation. This article reports a size-exclusion chromatography (SEC) method that permits the high-resolution analysis of LNP size distribution in its native solution condition. When coupled with multiple detection systems including UV-vis, multi-angle light scattering, and refractive index, on-line characterization of the distributions in size, molecular weight, and siRNA cargo loading of LNPs could be achieved. Six LNPs with sizes in the rang of 60-140 nm were evaluated and it was found that the SEC separation is efficient, highly reproducible, and can be broadly applied to a diverse range of LNPs. A comparison between the current SEC method and asymmetric field flow fractionation (FFF) shows that the current method provides similar size distribution results on LNPs compared to FFF. Two representative LNPs with similar bulk properties were evaluated in-depth using the SEC method along with two other sizing techniques-dynamic light scattering and cryo-TEM. Profound differences in batch polydispersity were observed between them. Despite the similarity in the particle assembly process, it was found that one LNP (A) possessed a narrow size and molecular weight distribution while the other (B) was polydisperse. The present results suggest that LNP drug products are highly complex and diverse in nature, and care should be taken in examining and understanding them to ensure quality and consistency. The method developed here can not only serve as a method for understanding LNP product property, permitting control on product quality, but also could serve as a potential manufacturing method for product purification. Understandings obtained in this work can help to facilitate the development of LNPs as a well-defined pharmaceutical product.
