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

项目摘要 mRNA疫苗已获得针对Covid-19的紧急授权批准，并正在开发用于 其他几种传染病。这些疫苗中的mRNA编码的免疫原是在脂质内递送的 纳米颗粒（LNP）包含四种脂质，最重要的是负责 通过质子化胺基团从细胞内核内体释放mRNA， 内体膜临床研究表明，这些疫苗对94%以上的患者非常有效 保护免受SARS-CoV-2感染。尽管针对SARS的mRNA LNP疫苗的成功开发- COV-2构成了一种新的医学模式的重大突破，它也突出了几个悬而未决的问题。 围绕该平台的问题需要紧急调查，包括：1）关键特征是什么 mRNA LNP制造需要尊重创造有效的系统？2)什么是功能性 通过体外翻译（IVT）或降解产生的截短mRNA转录物的后果以及如何 是否可以通过IVT优化将其最小化？3)是什么原因导致mRNA LNP降解， 如何测量和最大限度地减少这一点？ 我们最近发现了一种新的方法来合理地设计可电离的脂质，以增加效力。我们也 发现了一种新的制造方法来组装mRNA LNP，使得相同的脂质和mRNA 组件产生更有效和更有力的递送系统。这使得更多的mRNA在 LNP被翻译，从而减少剂量和反应原性，并增加可以被翻译的人数。 接种相同数量的疫苗。我们已经开始了研究， LNP的稳定性可能会影响这些疫苗的质量和性能。在本提案中，我们将执行 研究，以进一步了解mRNA LNPs是如何组装和制造在不同的 生产规模从实验室规模到商业规模。缺乏这方面的公共信息是一个问题。 这是改进和扩大这种新疫苗模式使用的主要障碍。我们还将阐明 mRNA原料药中不同转录物异质性的存在和重要性，以及 异质性会影响产品性能。最后，mRNA LNP在制造过程中的稳定性 和储存将进行研究，以了解造成生物活性和结构损失的降解机制 mRNA和脂质的完整性，如何测量这些特性，并开发具有延长 与现有产品相比，

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.