Manufacturing and Characterization of Potent mRNA Lipid Nanoparticle Vaccines at Multiple Scales

项目总结 信使核糖核酸疫苗已获得新冠肺炎的紧急授权批准，正在为 其他几种传染病。这些疫苗中的信使核糖核酸编码的免疫原在脂类中传递。 由四种脂类组成的纳米颗粒(LNP)，其中最重要的是负责 通过与细胞内内体相互作用的胺基的质子化，释放细胞内内体的mRNA 内胚体膜。临床研究表明，这些疫苗对94%以上的患者非常有效 免受SARS-CoV-2感染。尽管针对SARS的mRNA LNP疫苗的成功开发- CoV-2是一种新的医疗模式的重大突破，它也突出了几个悬而未决的问题 围绕该平台需要紧急调查的问题包括：1)关键功能有哪些 需要尊重的mRNALNP制造，以创建有效的系统？2)什么是功能性的 体外翻译(IVT)或降解产生的截短mRNA转录本的后果及其方式 能否通过IVT优化将其降至最低？3)是什么导致了在IVT过程中mRNA LNPs的降解 制造和储存，如何衡量和最小化这一点？ 我们最近发现了一种合理设计可电离脂质以提高效力的新工艺。我们也 发现了一种新的制造工艺来组装信使核糖核酸，使相同的脂质和信使核糖核酸 组件可以产生更高效、更强大的传输系统。这允许更多的信使核糖核酸 LNP将被翻译，从而减少剂量和反应性，并增加可以 接种相同数量的疫苗。我们已经开始了对mrna的特性和理解的研究。 LNP的稳定性会影响这些疫苗的质量和性能。在这份提案中，我们将执行 进一步了解mRNA LNPs是如何在不同的环境中组装和制造的研究 制造规模从实验室规模到商业规模。这一领域公共信息的缺乏是一个 这是改进和扩大这一新疫苗模式使用的主要障碍。我们还将介绍 不同转录本在信使核糖核酸药物物质中的异质性的存在和重要性，以及如何 异质性可能会影响产品性能。最后，mRNA LNPs在生产过程中的稳定性 并将对储存进行研究，以了解导致生物活性和结构丧失的降解机制 信使核糖核酸和脂质的完整性，如何测量这些特性，并开发已扩展的配方 与当前产品相比的稳定性概况。

PROJECT SUMMARY mRNA vaccines have received emergency authorization approval for Covid-19 and are under development for several other infectious diseases. The mRNA-encoded immunogen in these vaccines is delivered inside a lipid nanoparticle (LNP) that comprises four lipids, the most important being the ionizable lipid that is responsible for releasing the mRNA from intracellular endosomes through protonation of amine groups that interact with the endosomal membrane. Clinical studies showed these vaccines are highly efficacious with over 94% of patients protected from SARS-CoV-2 infection. Although the successful development of mRNA LNP vaccines for SARS- Cov-2 constitutes a major breakthrough for a novel medical modality, it has also highlighted several unanswered questions surrounding this platform that require urgent investigation including : 1) What are the critical features of mRNA LNP manufacturing that need to be respected to create potent systems ? 2) What is functional consequence of truncated mRNA transcripts produced by in vitro translation (IVT) or by degradation and how can they be minimized through IVT optimization ? 3) What causes degradation of mRNA LNPs during manufacturing and storage and how can this be measured and minimized ? We recently discovered a new process to rationally design ionizable lipids for increased potency. We also discovered a novel manufacturing process to assemble the mRNA LNP such that the same lipid and mRNA components produce a much more efficient and potent delivery system. This allows more of the mRNA in the LNP to be translated thereby reducing dose and reactogenicity and increasing the number of people that can be vaccinated with the same quantity of vaccine. We have initiated studies to characterize and understand mRNA LNP stability that can affect the quality and performance of these vaccines. In this proposal we will perform studies to further the understanding of how mRNA LNPs are assembled and manufactured at different manufacturing scales from laboratory scale to commercial scale. The lack of public information in this area is a major impediment to improving and broadening the use of this new vaccine modality. We will also illuminate the presence and importance of the heterogeneity of different transcripts in the mRNA drug substance and how that heterogeneity could impact product performance. And finally, the stability of mRNA LNPs during manufacturing and storage will be studied to understand the degradation mechanisms causing loss of bioactivity and structural integrity of the mRNA and lipids, how to measure these properties, and develop formulations that have extended stability profiles compared to current products.