信使RNA(mRNA)疫苗和疗法的发展需要生产具有特定关键质量属性(CQAs)的高质量体外转录mRNA药物。与线性DNA模板的均匀性密切相关。超螺旋质粒DNA是线性DNA模板的前体,超螺旋DNA百分比通常被认为是线性DNA模板制造过程中的关键过程控制(IPC)。在这项研究中,我们研究了超螺旋DNA百分比对关键mRNACQAs的影响,包括纯度,封顶效率,双链RNA(dsRNA),和聚(A)尾的分布。我们的发现揭示了超螺旋DNA百分比对mRNA纯度和体外转录产量的显着影响。值得注意的是,我们观察到对mRNA纯度的影响可以通过oligo-dT色谱来减轻,在一定程度上减轻了DNA超螺旋百分比的狭窄范围。总的来说,这项研究为DNA模板化学的IPC策略提供了有价值的见解,制造,和控制(CMC)和mRNA药物的过程开发。
The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality in vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.