Combined RNA Production and Purification Reagents Using Liquid-Liquid Phase Separating Biopolymers

项目摘要 这个I期SBIR项目的目标是开发一种新的纯化方法， 在一些实施方案中，本发明涉及基于转录（IVT）的RNA制造以用作治疗剂或疫苗。随着RNA变得越来越 通常用于制药产品，制造方法必须提高其可扩展性，速度， 成本效益。RNA制造中有几个痛点。首先，目前的亲和树脂具有非常大的 低容量-仅约2mg RNA/mL-需要商业规模的大体积树脂。这些树脂 每升的价格超过25，000美元，使得商业规模生产极其昂贵。第二，5 '封端 的RNA-在体内功能所必需的-和随后的任何残留的脱帽RNA的去除是一个主要的 RNA生产的瓶颈。Isolere Bio，Inc.专门设计创新的净化解决方案， 联合收割机结合了与弹性蛋白样多肽（ELP）基因融合的蛋白质亲和标签。该试剂能够 温度或盐控制的相分离，使捕获特定的目标，而不管其 复杂性或其他杂质的存在。我们将开发一种新的生物聚合物组合，称为 在此称为IVTag ™，其能够同时将RNA隔离成浓缩的纯液滴， 转录本的甲基化。将开发两种ELP融合蛋白：一种将含有RNA结合基序， 第二种是O ′-甲基转移酶（O ′-MTase），负责RNA的5 ′-加帽。然后我们将 开发平台方法，用于同时将RNA纯化成液滴并高效加帽 分离的RNA该I期项目旨在证明这种必需的IVT酶的相容性 并将其与Isolere的非色谱纯化方法相结合。该提案的主要活动包括 合成、纯化和测试每种生物聚合物融合物的功能，然后评估它们的功效， 串联应用，并将工艺效率与行业标准方法进行比较。如果成功， RNA微坩埚的功能将在第二阶段以T7聚合酶-ELP融合的形式开发 (for DNA模板的初始转录）和Poly-A聚合酶-ELP融合体（用于将3 'poly-A尾添加到 成绩单）。我们的愿景是最终在智能液滴中创建一个完全连续的RNA制造过程。 这些智能液滴有望支持一种简单、可扩展且具有成本效益的制造方式 微坩埚的工作流程能够执行一系列复杂的处理步骤。因为微- 坩埚的体积是独立的，消除了与亲和树脂相关的放大困难。此外，本发明还提供了一种方法， 随着Isolere技术的其他应用，包括抗体和病毒载体，ELP融合试剂 已经证明可容易地制造和重复使用以用于多个净化循环，从而降低商品成本 与亲和树脂相比有显著差异。的可扩展性、强大的性能和成本效益， 所提出的产品非常适合加速从实验室开发治疗性RNA候选物， 通过大规模生产商业产品进行大规模发现。

Project Summary The goal of this Phase I SBIR project is to develop a novel purification method that is compatible with in vitro transcription (IVT)-based manufacturing of RNA for use as a therapeutic or vaccine. As RNA becomes more commonly used for pharmaceutical products, manufacturing methods must improve their scalability, speed, and cost-effectiveness. There are several pain points in RNA manufacturing. First, current affinity resins have very low capacity —only ~2mg RNA per mL— necessitating large volumes of resin at commercial scale. These resins are priced at over $25,000 per liter, rendering commercial scale production extremely costly. Second, 5’ capping of RNA – essential for in vivo function – and the subsequent removal of any residual uncapped RNA is a major bottleneck for RNA production. Isolere Bio, Inc. specializes in designing innovative purification solutions that combine a protein affinity tag that is genetically fused to an elastin-like polypeptide (ELP). This reagent is capable of temperature or salt-controlled phase separation that enables capture of a specific target regardless of its complexity or the presence of other impurities. We will develop a novel combination of biopolymers, referred to here as IVTag™, that enables simultaneous RNA sequestration into concentrated, pure liquid droplets and methylation of transcripts. Two ELP fusion proteins will be developed: one will contain an RNA-binding motif and the second an O’-methyltransferase (O’-MTase) enzyme, responsible for 5’-capping of RNA. We will then develop platform methods for simultaneous purification of RNA into droplets and high efficiency capping of the sequestered RNA. This Phase I program is designed to demonstrate compatibility of this essential IVT enzyme and complexing it with Isolere’s non-chromatographic purification methods. Key activities of the proposal include synthesizing, purifying, and testing each biopolymer fusions’ functionality and then evaluating their efficacy when applied in tandem and comparing process efficiency to the industry standard method. If successful, additional functionality of the RNA micro-crucible will be developed in Phase II in the form of a T7 polymerase-ELP fusion (for initial transcription of a DNA template) and a Poly-A polymerase-ELP fusion (for adding a 3’ poly-A tail to transcripts). Our vision is to ultimately create a fully continuous RNA manufacturing process in smart droplets. These smart droplets are expected to support a straightforward, scalable, and cost-effective manufacturing workflow with micro-crucibles capable of carrying out a complex series of processing steps. Because the micro- crucible is volume independent, the scale-up difficulties associated with affinity resins are eliminated. In addition, with other applications of Isolere’s technology, including to antibodies and viral vectors, the ELP fusion reagents have proven to be easily manufacturable and re-usable for multiple purification cycles, reducing costs of goods significantly compared to affinity resins. The scalability, robust performance, and cost-effectiveness of the proposed product is an excellent fit for accelerating the development of therapeutic RNA candidates from bench scale discovery through large-scale manufacturing of commercial products.