LentiTag: A novel approach to the efficient manufacturing of active lentiviral vectors

LentiTag：一种高效制造活性慢病毒载体的新方法

• 批准号: 10384822
• 负责人: Kelli Michelle Luginbuhl
• 金额: $ 25.56万
• 依托单位: ISOLERE BIO, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384822
• 关键词: Address; Affinity; Anions; Attention; Behavior; Binding; Binding Proteins; Biological Assay; Biopolymers; Buffers; Cell Culture Techniques; Cell Therapy; Cells; Centrifugation; Chimeric Proteins; Chromatography; Clinic; Clinical; Clinical Trials; Dependence; Development; Diffuse; Disease; Escherichia coli; Exposure to; Filtration; Genetic Diseases; Glycoproteins; Goals; Harvest; Hydration status; Industry; Industry Standard; Ion-Exchange Chromatography Procedure; Jurkat Cells; Lentivirus; Lentivirus Vector; Liquid substance; Manufactured Supplies; Methods; Nature; Oncology; Outcome; Patients; Phase; Plant Resins; Process; Production; Property; Proteins; Reagent; Recombinants; Recovery; Research Personnel; Side; Small Business Innovation Research Grant; Sodium Chloride; Specificity; Suspensions; Technology; Temperature; Testing; Time; Translating; Vesicular stomatitis Indiana virus; Viral Vector; Virus; Work; base; chimeric antigen receptor; chimeric antigen receptor T cells; commercialization; cost; density; design; gene therapy; genetic payload; high throughput screening; improved; innovation; light scattering; new technology; novel; novel strategies; operation; personalized medicine; prevent; scale up; success; therapeutic development; transgene delivery; vector; vector vaccine; virus envelope

PROJECT SUMMARY Cell and gene therapies are garnering attention for their remarkable clinical outcomes and their promise to treat diseases previously considered uncurable. Lentiviral vectors (LVs) are used in over one third of applications in cell and gene therapies.2 As with most viral vector and vaccine manufacturing, LVs are challenging and expensive to manufacture and there are no methods in commercial use that offer specificity, high yield, and scalability. Current manufacturing relies on anion exchange chromatography, which (1) has poor capacities for large viruses that cannot effectively diffuse into small resin pores, (2) necessitates additional purification steps because its lack of LV specificity causes contaminant co-purification, and (3) requires harsh elution conditions that result in low infectious LV recoveries of only ~50%.15, 1 In this Phase I SBIR proposal, Isolere Bio will develop a fusion protein reagent comprised of an affinity domain that is specific for LV and a proprietary biopolymer domain that has robust and precisely tunable liquid-liquid phase separation behavior. The LentiTag™ reagent will capture LVs in solution with high specificity and then efficiently sequester them into phase separated droplets on command with a simple environmental trigger — an incremental adjustment in salt. These droplets, highly pure and concentrated, are easily separated from all excluded contaminants with tangential flow filtration (TFF), a unit operation that scales up simply and enables high volumetric throughput. Once host cell proteins and other cellular contaminants have been washed away, the LV can be gently recovered from the droplets with an elution buffer that disrupts the affinity interaction at near-neutral pH. To demonstrate LentiTag™’s technical feasibility, Isolere will first design, produce, and characterize an LV-specific capture protein (LCP). The LCP binds the most common LV envelope glycoprotein and phase separates at ambient temperature with a small increase in salt (<0.35M NaCl) that is tolerated by labile LVs. Having defined optimal capture conditions, we will next perform high-throughput screening of LV-compatible elution buffers and then optimize key filtration process parameters, including pore size and permeate flow rate. We will perform a side-by-side comparison of LentiTag™ to the standard industry process, quantifying final LV concentration, infective titer, yield, purity, and total processing time. Last, because the LCP is well-hydrated and sterically occluding in its soluble state, we also hypothesize that it can confer protective and stabilizing effects to LVs, which are prone to progressive activity loss and aggregation during storage. Our final aim is to explore the impact of our reagent on LV stability, both during upstream production (preventing host cell auto-transduction) and as an additive to formulated LV stored at temperatures ranging from -70ºC to 37 ºC. The LentiTag™ technology will provide a scalable platform for LV manufacturing that will help to both democratize and accelerate the discovery, development, and commercialization of cell and gene therapies around the globe.

项目摘要 细胞和基因疗法引起了人们对其显着临床结果的关注以及他们对治疗的承诺 疾病以前被认为是无关的。慢病毒载体（LVS）用于超过三分之一的应用 细胞和基因疗法。2与大多数病毒载体和疫苗制造一样，LV是挑战，并且 制造昂贵，商业用途没有提供特异性，高收益和 可伸缩性。当前的制造依赖于阴离子交换色谱法，（1）的能力较差 无法有效扩散成小树脂孔的大型病毒，（2）必要的其他纯化步骤 因为它缺乏LV特异性会导致污染物的共纯化，并且（3）需要危害洗脱条件 这导致感染力低下的LV回收率仅为50％.15，1在这阶段I SBIR提案中，Iselere Bio将会发展 融合蛋白试剂完成了特定于LV和专有生物聚合物的亲和力结构域 具有鲁棒且精确的液态液相分离行为的结构域。 Lentitag™试剂 将捕获具有高特异性的溶液中的LV，然后有效地将它们隔离为相分离的液滴 用简单的环境触发器进行命令 - 盐的增量调整。这些液滴很高 纯和集中，很容易与所有排除的具有切向流量过滤（TFF）的排除污染物分离， 单位操作简单地扩展并实现高体积吞吐量。一旦宿主细胞蛋白和其他 细胞污染物已被洗掉，可以从液滴中轻轻回收LV 缓冲液会破坏接近中性pH的亲和力相互作用。为了展示Lentitag™的技术可行性， Inlyere将首先设计，生产和表征LV特异性捕获蛋白（LCP）。 LCP结合最多 常见的LV包膜糖蛋白和相位在环境温度下分离，盐较小 （<0.35亿NaCl），通过不稳定的LV耐受。在定义的最佳捕获条件下，我们接下来会执行 LV兼容洗脱缓冲液的高通量筛选，然后优化密钥过滤过程参数， 包括孔径和渗透流速。我们将对Lentitag™进行并排比较 标准行业流程，量化最终LV浓度，感染性滴度，产量，纯度和总处理 时间。最后，因为LCP的水分良好，并且在其固态处于空间遮挡，我们也假设 它可以将受保护和稳定效应与LVS稳定，这很容易进行渐进的活动损失和 存储期间的聚合。我们的最终目的是探索我们的试剂对LV稳定性的影响 上游生产（防止宿主细胞自动转移），作为存储在 温度范围从-70ºC到37ºC。 Lentitag™技术将为LV提供可扩展的平台 制造业将有助于使发现，发展和加速 全球细胞和基因疗法的商业化。