Department of Pharmaceutical Sciences, Merck Research Laboratories, Merck & Co, Inc, West Point, Pennsylvania 19486, United States.
Mol Pharm. 2013 Jan 7;10(1):397-405. doi: 10.1021/mp3005337. Epub 2012 Dec 19.
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.
当开发基于脂质纳米颗粒 (LNP) 的小干扰 RNA (siRNA) 治疗药物时，首要考虑的因素是制剂多分散性或异质性。药物批次中物理化学性质的异质性水平可能会极大地影响生物性能、质量以及制造商控制和重现制剂的能力。本文通过使用基于 Sephacryl S-1000 的大小排阻色谱 (SEC) 方法进行制备规模分级，研究了含有 siRNA 的 LNP 的大小、组成和体外性能的异质性。首先，通过 SEC 方法评估了 8 种大小在 60-190nm 范围内的 LNPs 的多分散性特征，结果发现 60-150nm 的 LNPs 可以很好地分离。对具有相似体相性质的两种 LNP（LNP A 和 LNP B）进行分级，并深入研究各馏分在大小、组成和体外沉默以及细胞毒性方面是否存在多分散性。结果表明，LNP A 经过半制备 SEC 分级后被认为是单分散的，其大小、化学组成、体外沉默活性和细胞毒性在各馏分中均相似。因此，LNP A 代表了一种相对均匀的制剂，在其药物开发中面临的挑战较小。相比之下，LNP B 馏分在大小分布上表现出显著的多分散性。有趣的是，LNP B SEC 馏分还表现出明显的组成变化（例如，N/P 比差异 5 倍，脂质组成差异 3 倍），以及体外沉默活性差异高达 40 倍。还讨论了 LNP 大小和制剂组成对体外性能的影响。目前的结果表明，LNP 基于 siRNA 药物产品中存在复杂性和异质性的可能性。这强调了需要有工具来对 LNP 制剂进行详细表征。这种能力与追求改进的制剂和工艺设计相结合，可以更轻松地开发更高质量的基于 LNP 的 siRNA 药物。
