Improving mRNA vaccine manufacture using RNA sequencing

The manufacture mRNA vaccines needed to manufacture billions of doses at sufficient quality during the COVID19 pandemic. The removal of contaminating RNAs and the inclusion of modified nucleotides reduces the innate immune response and improves the translation and performance of mRNA vaccines and therapies. Here we validate the use of nanopore sequencing during the manufacture and analysis of mRNA vaccines. Using a modified protocol and software toolkit, we show how nanopore sequencing enables quantitative analysis of mRNA vaccine sequence, length and purity, including off target RNA impurities. These analyses inform best practise approaches for designing mRNA sequences, optimise manufacturing processes and benchmark purification methods. We also perform direct RNA sequencing of mRNA vaccines to measure pseudouridines incorporation errors and 5 prime capping. Together this shows how nanopore sequencing can be used during mRNA manufacture to validate batches, monitor quality and provide a detailed characterisation of the final mRNA vaccine product.

在新冠疫情（COVID-19）大流行期间，需以足够高的质量规模化生产数十亿剂信使核糖核酸（mRNA）疫苗。去除污染性RNA并添加修饰核苷酸（modified nucleotides），可降低先天免疫应答，同时优化mRNA疫苗与疗法的翻译效率及应用性能。本研究验证了纳米孔测序（nanopore sequencing）在mRNA疫苗生产与分析流程中的应用价值。通过改良的实验方案（protocol）与软件工具包（software toolkit），我们证实纳米孔测序可实现对mRNA疫苗的序列、长度与纯度的定量分析，包括脱靶RNA杂质（off target RNA impurities）的检测。此类分析可为mRNA序列设计的最佳实践提供指导，优化生产工艺，并为纯化方法建立性能基准。我们还对mRNA疫苗开展直接RNA测序（direct RNA sequencing），以检测假尿苷（pseudouridines）掺入误差与5'端加帽（5 prime capping）情况。综上，本研究证实纳米孔测序可应用于mRNA疫苗的生产环节，用于批次验证、质量监控，并可对最终mRNA疫苗产品进行全面的特性表征。