最近批准信使核糖核酸(mRNA)作为对抗COVID-19大流行的疫苗是一个科学转折点。今天,mRNA的适用性正在被证明超出传染病,例如在癌症免疫疗法中,蛋白质替代疗法和基因编辑。mRNA由线性DNA模板通过体外转录(IVT)产生,并在3'和5'末端修饰以提高翻译效率和稳定性。共存的杂质,如RNA片段和双链RNA(dsRNA),在其他人中,会极大地影响mRNA的质量和功效。在这项研究中,评估大小排阻色谱法(SEC)对IVT-mRNA的表征。流动相组成(离子强度和有机改性剂)的影响,pH值,柱温和孔径(300µ,1000贝达,描述了大小不同的IVT-mRNA的分离性能和结构完整性。非复制,自扩增(saRNA),温度下降,和核糖核酸酶(RNase)消化的mRNA,后者表征3'聚(A)尾,包括在研究中。超越紫外线(UV)检测,进行折射率(RI)和多角度光散射(MALS)检测以准确测定mRNA的分子量(MW)。最后,引入质量光度法作为在天然条件下研究mRNA的补充方法。
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.