Developing a rapid quality control and long-term stability assay for RNA vaccine candidates

针对SARS-Cov2的RNA疫苗已经显示出巨大的前景，临床试验的早期结果表明&gt；90%的保护作用被赋予。这些疫苗的RNA成分非常长，最多可达10,000个核苷酸，这带来了分析挑战，因为凝胶电泳等标准方法不足以检测链长的微小差异。此外，检测高效翻译所必需的5‘帽的存在的分析依赖于缓慢而费力的方法。因此，需要开发新的分析技术，应用于RNA疫苗的质量控制/质量保证，以支持疫苗的制造和储存期间长期稳定性的评估。我们先前已经证明了一种新的RNA分析的概念验证，该方法能够一端捕获分子，然后专门针对另一端进行探测，只有当RNA分子完整并包含5‘帽时才能产生可测量的信号。然而，当应用于疫苗等长RNA分子时，由于最初捕获步骤中的空间位阻，该分析失去了敏感性。因此，分析需要高浓度的RNA，这可能会限制该分析的整体应用。在这个项目中，我们的目标是探索一系列旨在最大化RNA捕获的干预措施。我们将对项目中的三种策略进行比较。首先，我们将使用统计实验设计，通过改变所涉及的不同分子的浓度来优化我们现有的检测，以最大化信噪比。其次，我们将尝试逆转检测，以便我们通过5‘端捕获RNA分子，并在相反的一端进行探针。最后，我们将研究是否有可能完全省略捕获步骤，形成一个连接RNA分子两端的复合体，从而导致荧光信号。作为概念验证，我们将把这种分析应用于目前正在进行临床试验的帝国理工学院Sarna候选疫苗。为了建立在这项概念验证研究的基础上，我们的目标是与广泛的利益攸关方接触，使疫苗制造商能够采用该方法，并将寻求监管部门的批准，使该检测方法能够用于批量释放测试。

RNA vaccines against SARS-Cov2 have shown great promise with early results from clinical trials indicating >90% protection conferred. The RNA component of these vaccines is very long, up to 10,000 nucleotides, which introduces analytical challenges as standard methods such as gel electrophoresis are not sufficient to detect small differences in chain length. In addition, assays to detect the presence of the 5' cap that is necessary for efficient translation rely on slow and laborious methods. Thus, there is a need to develop new analytical technologies that can be applied to the QC/QA of RNA vaccines to support both manufacturing and the assessment of the long-term stability of vaccines during storage.We have previously demonstrated a proof-of-concept for a new RNA assay that is able to capture the molecule by one end and then probe specifically for the other end, leading to a measurable signal only when the RNA molecule is intact and contains a 5' cap. However, when applied to long RNA molecules such as vaccines, the assay loses sensitivity due to steric hindrance in the initial capture step. Therefore, high concentrations of RNA are required for analysis, which might limit the overall application of the assay. In this project, we aim to explore a series of interventions designed to maximise RNA capture. We will compare three strategies in the project. First, we will use statistical design of experiments to optimise our existing assay by varying the concentrations of the different molecules involved to maximise the signal-to-noise ratio. Second, we will try reversing the assay so that we capture the RNA molecule by the 5' cap and probe for the opposite end. Finally, we will examine whether it is possible to omit the capture step entirely and form a complex that bridges both ends of the RNA molecule leading to a fluorescence signal.As a proof-of-concept we will apply the assay to the Imperial College saRNA vaccine candidate that is currently undergoing clinical trials. To build on this proof-of-concept study, we aim to engage with a broad variety of stakeholders to enable uptake by vaccine manufacturers and will seek regulatory approval to enable the assay to be used for batch release testing.