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 细胞和基因疗法因其显著的临床效果和治疗前景而备受关注 以前认为无法治愈的疾病。慢病毒载体（LV）用于超过三分之一的应用， 细胞和基因治疗。2与大多数病毒载体和疫苗制造一样，LV具有挑战性， 制造昂贵，并且在商业应用中没有提供特异性、高产率和 可伸缩性目前的生产依赖于阴离子交换色谱，其（1）具有差的分离能力， 不能有效扩散到小树脂孔中的大病毒，（2）需要额外的纯化步骤 因为其缺乏LV特异性导致污染物共纯化，和（3）需要苛刻的洗脱条件 这导致感染性LV回收率较低，仅为~ 50%。15，1在本阶段SBIR提案中，Isolere Bio将开发 融合蛋白试剂，由特异性针对LV的亲和结构域和专有生物聚合物组成 该领域具有鲁棒且精确可调的液-液相分离行为。LentiTag™试剂 将以高特异性捕获溶液中的LV，然后将它们有效地隔离到相分离的液滴中 通过一个简单的环境触发-盐的增量调整。这些飞沫，高度 纯的和浓缩的，很容易用切向流过滤（TFF）从所有排除的污染物中分离出来， 单元操作可简单地按比例放大并实现高体积吞吐量。一旦宿主细胞蛋白质和其他 当细胞污染物被洗掉时，可以用洗脱液从液滴中温和地回收LV 在接近中性的pH下破坏亲和相互作用的缓冲液。为了证明LentiTag™的技术可行性， Isolere将首先设计，生产和表征LV特异性捕获蛋白（LCP）。LCP结合最多 一种常见的LV包膜糖蛋白，在环境温度下发生相分离，盐浓度略有增加 （<0.35M NaCl），其被不稳定LV耐受。在定义了最佳捕获条件之后，我们接下来将执行 高通量筛选LV兼容洗脱缓冲液，然后优化关键过滤工艺参数， 包括孔径和渗透流速。我们将对LentiTag™和 标准工业过程，定量最终LV浓度、感染滴度、产量、纯度和总处理 时间最后，由于LCP在其可溶状态下水化良好且空间闭塞，我们还假设 它可以赋予LV保护和稳定作用，LV易于进行性活动丧失， 在存储期间聚集。我们的最终目的是探索我们的试剂对LV稳定性的影响，无论是在 上游生产（防止宿主细胞自动转导），并作为配制的LV的添加剂， 温度范围从-70播放C到37 ºC。LentiTag™技术将为LV提供可扩展的平台 制造业，这将有助于民主化和加速发现，发展， 细胞和基因疗法在地球仪的商业化。