最近批准信使核糖核酸 (mRNA) 作为对抗 COVID-19 大流行的疫苗，这是一个科学转折点。如今，mRNA 的适用性已不仅仅局限于传染病，例如在癌症免疫疗法、蛋白质替代疗法和基因编辑中。 mRNA 由线性 DNA 模板通过体外转录 (IVT) 产生，并在 3' 和 5' 末端进行修饰，以提高翻译效率和稳定性。 RNA 片段和双链 RNA (dsRNA) 等共存杂质可能会极大地影响 mRNA 的质量和功效。在本研究中，评估了尺寸排阻色谱 (SEC) 对 IVT-mRNA 的表征。描述了流动相组成（离子强度和有机改性剂）、pH、柱温和孔径（300 Å、1000 Å 和 2000 Å）对不同尺寸的 IVT-mRNA 的分离性能和结构完整性的影响。该研究包括非复制、自扩增 (saRNA)、温度降解和核糖核酸酶 (RNase) 消化的 mRNA，后者用于表征 3' Poly(A) 尾。除了紫外线 (UV) 检测之外，还实施了折射率 (RI) 和多角度光散射 (MALS) 检测，以准确确定 mRNA 的分子量 (MW)。最后，引入质量光度测定法作为在天然条件下研究 mRNA 的补充方法。版权所有 © 2024。由 Elsevier B.V. 出版。

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.Copyright © 2024. Published by Elsevier B.V.