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

项目摘要 基因疗法仍然是广泛的难治性疾病的有希望的候选人，但其复杂性 使得传统的生物制药制造过程效率低、成本高且不切实际。鉴于 单剂量的AAV递送的基因治疗可能需要从60 μ L培养基中纯化病毒，或者 此外，迫切需要以可扩展的、高吞吐量的和低成本的方式来简化该过程， 有效的解决方案。超离心法和层析法是AAV最常用的分离方法， 纯化，目前在商业生产中没有“金标准”方法。当前的这些局限性 方法促使Isolere Bio，Inc.开发创新的病毒纯化平台：ViraTag™。 ViraTag™涉及两个连续步骤。首先，病毒被定制蛋白试剂亲和捕获， 使用者通过简单的环境变化，例如添加盐， 或加热。病毒颗粒与宿主细胞蛋白质和其他蛋白质一起被隔离在与水不混溶的液滴中。 残留在水相中的细胞污染物。其次，一旦污染物被冲走， 通过降低pH从液滴中提取AAV，这将它们从捕获试剂中释放出来。 ViraTag™解决了理想纯化工艺的关键要求，包括：1）线性可扩展性， 容纳103至105 L的输入培养基; 2）温和的洗脱条件，以保留载体功能和治疗效果 有效性; 3）验证了与所用自动化下游纯化仪器和工作流程的兼容性 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潜力 从空衣壳中分离出来在SBIR快速通道提案的第一阶段，我们将设计一种新的ViraTag™试剂 能够亲和捕获和相分离AAV颗粒，并评估其在AAV纯化中的效率 从细胞培养基中。我们还将评估这种新的亲和捕获试剂区分“全”的能力。 与“空”衣壳相比，这是一种迫切需要的能力，目前只有通过超浓缩方法才能实现， 不能扩大规模。第二阶段将重点关注ViraTag™生产的规模扩大，通过开发工作流程和 各种应用规模的产品外形规格，并根据当前的标准对最终产品的纯度进行基准测试 方法.第二阶段还将包括由具有代表性的实验室对工艺进行外部验证。 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.