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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要细胞和基因疗法因其卓越的临床效果和治疗前景而受到关注此前，超过三分之一的疾病都使用慢病毒载体（LV）。细胞和基因疗法。2 与大多数病毒载体和疫苗制造一样，LV 具有挑战性，并且制造成本昂贵，并且没有商业用途的方法能够提供特异性、高产率和当前的制造依赖于阴离子交换色谱，其 (1) 的能力较差。无法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它缺乏 LV 特异性，导致污染物共纯化，并且 (3) 需要苛刻的洗脱条件导致传染性 LV 回收率较低，仅为约 50%.15, 1 在这一 I 期 SBIR 提案中，Isolere Bio 将开发一种融合蛋白试剂，由 LV 特异性亲和结构域和专有生物聚合物组成具有稳健且精确可调的液-液相分离行为的 LentiTag™ 试剂。将以高特异性捕获溶液中的 LV，然后有效地将它们隔离到相分离的液滴中通过简单的环境触发命令——盐的增量调整。纯净且浓缩，可通过切向流过滤 (TFF) 轻松与所有排除的污染物分离，一种可简单放大并实现高体积通量的单元操作。细胞污染物已被冲走，可以通过洗脱从液滴中轻轻回收 LV 在接近中性 pH 值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。常见的 LV 包膜糖蛋白，并在环境温度下随着盐的少量增加而发生相分离 (<0.35M NaCl) 是不稳定 LV 所耐受的。有了最佳捕获条件，我们接下来将执行。高通量筛选 LV 兼容的洗脱缓冲液，然后优化关键过滤工艺参数，我们将对 LentiTag™ 与 LentiTag™ 进行并排比较。标准工业流程，量化最终 LV 浓度、感染滴度、产量、纯度和总处理量最后，由于 LCP 在可溶状态下具有良好的水合性和空间闭锁性，因此我们还捕获到了这种情况。它可以赋予 LV 保护和稳定作用，而 LV 容易逐渐丧失活性并赋予我们的最终目标是探索我们的试剂对 LV 稳定性的影响，无论是在储存期间还是在储存期间。上游生产（防止宿主细胞自转导）并作为储存在配制的 LV 的添加剂 LentiTag™ 技术将为 LV 提供可扩展的平台，温度范围为 -70°C 至 37°C。制造业将有助于民主化并加速发现、开发和全球细胞和基因疗法的商业化。