ViraTag: A Scalable Purification Technology for Adeno-Associated Virus Gene Therapy Vectors

项目总结 基因疗法仍然是一系列顽固疾病的有希望的候选者，然而它们的复杂性。 使传统的生物制药生产程序效率低、成本高、不切实际。考虑到一个 单剂甲型肝炎病毒基因治疗可能需要从60 L或 此外，还有一种迫切且未得到满足的需求，即通过可扩展、高吞吐量和成本- 有效的解决方案。而超速离心法和层析法是检测AAV最常用的方法 在提纯方面，目前还没有商业化生产的“黄金标准”方法。电流的这些限制 这些方法促使Isolere生物公司开发了一种创新的病毒纯化平台：ViraTag™。 ViraTag™包括两个连续的步骤。首先，病毒被定制的蛋白质试剂亲和捕获，并且 用户通过简单的环境变化，例如添加盐，来触发液-液相变 或者热度。病毒颗粒被隔离在不溶于水的液滴中，与宿主细胞蛋白质和其他 留在水相中的细胞污染物。第二，一旦污染物被冲走， 通过降低pH从液滴中提取AAV，从而将它们从捕捉剂中释放出来。 ViraTag™解决了理想纯化过程的关键要求，包括：1)线性可伸缩性 可容纳103-105个L的输入介质；2)温和的洗脱条件，保留向量功能和治疗作用 功效；3)与使用的自动化下游净化仪器和工作流程的兼容性验证 在生物制造中，以及4)分离含有治疗性核酸有效载荷的衣壳的可能性 从空壳中脱身。在SBIR快速通道计划的第一阶段，我们将设计一种新的病毒标签™试剂 能够对AAV颗粒进行亲和捕获和相分离，并评价其在AAV纯化中的效率 从细胞培养液中提取。我们还将评估这种新的亲和捕捉剂区分“Full”的能力。 相对于“空的”衣壳，一种目前急需的能力只有通过超速离心法才能实现， 不能放大。第二阶段将专注于扩大ViraTag™制造，方法是开发工作流程和 适用于不同应用规模的产品外形系数，并根据当前的情况对最终产品的纯度进行基准测试 方法：研究方法。第二阶段还将包括由以下实验室代表对该过程进行外部验证 产品的目标客户，从实验室规模的学术研究到大规模的生物制药。

PROJECT SUMMARY Gene therapies remain promising candidates for a broad range of intractable diseases, however their complexity renders traditional biopharmaceutical manufacturing procedures inefficient, costly, and impractical. Given that a single dose of an AAV-delivered gene therapy may require purification of virus from 60 L of culture media or more, there is an urgent and unmet need to streamline the process with a scalable, high throughput, and cost- effective solution. While ultracentrifugation and chromatography are the most common methods for AAV purification, there is currently no “gold standard” method in commercial production. These limitations of current methods have motivated Isolere Bio, Inc. to develop an innovative purification platform for viruses: ViraTag™. ViraTag™ involves two sequential steps. First, the viruses are affinity captured by a custom protein reagent and the user triggers a liquid-liquid phase transition by a simple environmental change, such as the addition of salt or heat. The virus particles are sequestered into water-immiscible droplets, with host cell proteins and other cellular contaminants left behind in the aqueous phase. Second, once contaminants have been washed away, the AAV are extracted from the droplets by lowering pH, which releases them from the capture reagent. ViraTag™ addresses key requirements for an ideal purification process, including: 1) linear scalability to accommodate 103 to 105 L of input media; 2) mild elution conditions to preserve vector function and therapeutic efficacy; 3) validated compatibility with automated downstream purification instrumentation and workflows used in biomanufacturing, and 4) the potential to isolate capsids containing therapeutic nucleic acid payloads away from empty capsids. During Phase I of this SBIR Fast track proposal, we will engineer a new ViraTag™ reagent capable of affinity capture and phase separation of AAV particles, and evaluate its efficiency in AAV purification from cell culture media. We will also evaluate the ability of this new affinity capture reagent to distinguish “full” versus “empty” capsids, an urgently needed capability currently only enabled by ultracentrifugation methods that cannot be scaled up. Phase II will focus on scale-up of ViraTag™ manufacturing, by developing workflows and product form factors for various application scales, and benchmarking the purity of final products against current methods. Phase II will also include external validation of the process by laboratories representative of the product’s target customers, ranging from bench-scale academic research to large-scale biopharma.