在COVID-19大流行之后,信使RNA(mRNA)彻底改变了传统的疫苗制造。随着越来越多的基于RNA的疗法,对这些分子有价值的新科学见解已经出现。一个令人着迷的研究领域是在体外转录(IVT)过程中双链RNA(dsRNA)的形成,这被认为是一种重要的杂质。因为它已被确定为细胞免疫应答途径的主要触发因素。因此,开发和优化用于去除该副产物的纯化工艺越来越重要。传统上,努力主要集中在通过层析分离进行IVT后的mRNA纯化,阴离子交换和反相色谱成为实现这一目的的有效工具。然而,据我们所知,线性化质粒质量的影响和意义尚未得到彻底研究。质粒的生产涉及细菌培养物的生长,细菌收获和裂解,和用于质粒DNA纯化的多个过滤步骤。这些分子固有的复杂性,连同其加工过程中涉及的众多纯化步骤,包括随后的线性化和较不发达的线性化质粒纯化技术,通常导致不一致的批次,对副产物如dsRNA的控制有限。本研究旨在证明用于线性化质粒的纯化过程如何影响dsRNA的形成。基于这两种方法纯化线性化质粒的几种技术,树脂过滤和色谱分离,已经研究过了。因此,我们已经优化了色谱方法,使用具有C4化学(丁基链位于颗粒表面)的整体柱纯化线性化质粒,已证明对各种大小的mRNA都是成功的。这种色谱分离有助于产生均匀的线性化质粒,导致在随后的IVT过程中dsRNA水平较低的mRNA批次。该发现揭示了dsRNA形成不仅受RNA聚合酶和IVT条件的影响,而且受线性化模板的质量的影响。结果表明,质粒杂质可通过提供可被转录成与mRNA分子退火的序列的额外模板而有助于dsRNA的产生。这突出了考虑与转录期间的dsRNA生成相关的质粒纯化质量的重要性。需要进一步的研究来充分理解质粒衍生的dsRNA的机制和含义。这一发现可能会转移mRNA疫苗生产的焦点,更加强调线性化质粒的纯化和潜在的节约,在某些情况下,IVT后的mRNA纯化步骤。
After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (
IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after
IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent
IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and
IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following
IVT.