Two imaged capillary isoelectric focusing methods were developed to provide insight into the quality and stability of messenger ribonucleic acid (mRNA) vaccines, specifically, mRNA encapsulated in lipid nanoparticles (LNPs). A variety of stressed and lipid composition-modified samples were measured and detected by their UV absorption. The results were supported by the data of an encapsulation assay and particle sizing. One method, using 9 M urea as an additive, shows two broad and jagged peaks in which the peak shape offers detailed information. The summed peak area of both peaks showed RSDs from 2% to 8% when one batch was measured in triplicate and apparently depends on the size of the LNPs. In the second method, a combination of 5.5 M urea and 2 M N-ethylurea was used. This method is characterized by a high repeatability of the isoelectric point (pI, <0.5%). The repeatable peak area (RSD of 2%–7%) correlates linearly with the mRNA content, which also applies to the first method, and added stress is evident by the change in pI and peak area. Furthermore, experiments with the addition of a fluorescent dye were performed (fluorescence detection), which tremendously increased the sensitivity of the methods. Both methods can be used to characterize the stability of mRNA-loaded LNPs, for example, when investigating various storage times at different temperatures and freeze–thaw cycles, as well as the ability of the methods to distinguish lipid compositions and measure batch-to-batch variability.

中文翻译：

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两种质量和稳定性表明 mRNA 疫苗成像 CIEF 方法的方法


开发了两种成像毛细管等电聚焦方法，以深入了解信使核糖核酸 (mRNA) 疫苗，特别是封装在脂质纳米粒子 (LNP) 中的 mRNA 的质量和稳定性。通过紫外吸收来测量和检测各种受压和脂质成分修饰的样品。结果得到了封装测定和粒度测定数据的支持。一种方法使用 9 M 尿素作为添加剂，显示两个宽且锯齿状的峰，其中峰形状提供了详细信息。当一批一式三份进行测量时，两个峰的总峰面积显示 RSD 为 2% 至 8%，并且显然取决于 LNP 的大小。在第二种方法中，使用5.5M尿素和2M N-乙基脲的组合。该方法的特点是等电点（p I 、<0 id=2>I 和峰面积）具有高重复性。此外，还进行了添加荧光染料的实验（荧光检测），大大提高了检测的灵敏度。这两种方法都可用于表征 mRNA 负载的 LNP 的稳定性，例如，在研究不同温度和冻融循环下的各种储存时间时，以及该方法区分脂质成分和测量批次的能力。 - 批次差异。