mRNA Center of Excellence, Sanofi, Waltham, Massachusetts 02451, United States.
Anal Chem. 2024 Apr 9;96(14):5570-5579. doi: 10.1021/acs.analchem.4c00031. Epub 2024 Mar 26.
Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect 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 subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl--glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl--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 topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were 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 topmost 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 subpopulations.
mRNA 脂质纳米粒（LNPs）的优化传递需要合理的设计和稳健的制剂工艺。mRNA-LNPs 的异质性程度不同会影响其物理和功能特性。鉴于 mRNA-LNPs 的复杂性，开发全面和正交的分析技术对于更好地理解这些制剂至关重要。为此，我们开发了一种稳健的基于超速离心的方法，用于基于密度分离 mRNA-LNP 的亚群。使用两种可离子化脂质 A 和 B，以及两种辅助脂质 1,2-二油酰基-sn-甘油-3-磷酸乙醇胺（DOPE）和 1,2-二油酰基-sn-甘油-3-磷酸乙醇胺（DEPE），以及胆固醇和 DMG-PEG-2K，合成了四种包封人红细胞生成素（hEPO）的 LNP 制剂，具有不同的功能。在蔗糖梯度上超速离心后，从最上面的低密度部分到越来越密集的底部部分，可以观察到整个梯度上出现明显的“馏分”模式，这些馏分被收集用于全面分析。亲代 LNPs、A-DOPE 和 B-DOPE 被分为三个基于密度的馏分，每个馏分在静脉和肌肉内给药途径后的 hEPO 表达差异显著。亲代 B-DEPE LNPs 分为两个基于密度的馏分，与下一个馏分相比，大多数有效载荷和脂质含量归因于最上面的馏分，表明存在一定程度的异质性，而亲代 A-DEPE LNPs 表现出显著的均一性，三个基于密度的馏分之间的体内效力、脂质数量和颗粒计数相当。这项研究首次证明了基于密度梯度的超速离心（DGC）在作为生物性能和物理化学特性的函数的亲代 mRNA-LNPs 及其亚群的异质性的头对头比较中的应用。
