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

针对SARS-Cov 2的RNA疫苗已经显示出巨大的前景，来自临床试验的早期结果表明赋予>90%的保护。这些疫苗的RNA组分非常长，高达10，000个核苷酸，这引入了分析挑战，因为标准方法如凝胶电泳不足以检测链长度的微小差异。此外，检测有效翻译所必需的5'帽的存在的测定依赖于缓慢且费力的方法。因此，有必要开发新的分析技术，可以应用于RNA疫苗的QC/QA，以支持疫苗在储存期间的生产和长期稳定性评估。我们以前已经证明了一种新的RNA检测方法的概念验证，该方法能够通过一端捕获分子，然后针对另一端进行特异性探针检测，导致只有当RNA分子是完整的并且含有5 ′帽时才有可测量的信号。然而，当应用于长RNA分子如疫苗时，由于初始捕获步骤中的空间位阻，该测定法失去了灵敏度。因此，分析需要高浓度的RNA，这可能限制测定的整体应用。在这个项目中，我们的目标是探索一系列旨在最大限度地捕获RNA的干预措施。我们将在项目中比较三种策略。首先，我们将使用实验的统计设计，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方法，以最大限度地提高信噪比。第二，我们将尝试逆转测定，以便我们通过5'帽捕获RNA分子，并将探针用于相对末端。最后，我们将研究是否有可能完全省略捕获步骤，并形成一个复合物，桥接RNA分子的两端，导致荧光信号。作为概念验证，我们将应用该检测方法对目前正在进行临床试验的帝国理工学院saRNA候选疫苗。为了建立在这个概念验证研究的基础上，我们的目标是与各种各样的利益相关者合作，使疫苗制造商能够采用，并将寻求监管部门的批准，使该检测方法能够用于批放行检测。

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.