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

项目总结 细胞和基因疗法因其显著的临床效果和治疗前景而受到关注。 以前被认为是无法治愈的疾病。慢病毒载体(LV)在超过三分之一的应用中使用 细胞和基因治疗2与大多数病毒载体和疫苗制造一样，LV具有挑战性和 制造成本高昂，目前还没有商业用途的方法提供特异性、高产率和 可扩展性。目前的生产依赖于阴离子交换层析，这(1)具有较差的能力 不能有效地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，以及(3)要求苛刻的洗脱条件 这将导致低感染性LV恢复率仅为~50%。15，1在此阶段的SBIR建议中，Isolere Bio将开发 一种融合蛋白试剂，由LV特异的亲和域和专有的生物聚合物组成 具有稳健且精确可调的液-液相分离行为的领域。LentiTag™试剂 将以高度的特异性捕获溶液中的LV，然后将它们有效地隔离成相分离的液滴 只需一个简单的环境触发器--盐分的增量调整--就可以了。这些水滴，高度 纯的和浓缩的，通过切向流过滤(TFF)很容易从所有排除的污染物中分离出来， 一种简单的单元操作，可实现高容量吞吐量。一旦宿主细胞蛋白质和其他 细胞污染物已被洗去，洗脱后可从液滴中轻轻回收LV 在近中性pH时破坏亲和相互作用的缓冲液。为了证明LentiTag™的S的技术可行性， Isolere将首先设计、生产和鉴定一种LV特异性捕获蛋白(LCP)。LCP绑定最多 常见的LV包膜糖蛋白和相在常温下分离，盐的含量略有增加 (&lt；0.35M氯化钠)，这是不稳定的LV所能容忍的。定义了最佳捕获条件后，接下来我们将执行 高通量筛选与LV兼容的洗脱缓冲液，然后优化关键的过滤工艺参数， 包括孔径大小和渗透流速。我们将执行LentiTag™与 标准工业流程，量化最终病毒浓度、感染滴度、产量、纯度和总加工 时间到了。最后，由于LCP在其可溶状态下是水合良好且空间封闭的，我们还假设 它可以赋予LV保护和稳定的作用，LV容易进行性活动丧失和 存储期间的聚合。我们的最终目标是探索我们的试剂对左心室稳定性的影响，在这两个过程中 上游生产(防止宿主细胞自动转导)，并作为配方LV的添加剂储存在 LentiTag™技术将为LV提供可扩展的平台 制造业将有助于民主化和加速发现、开发和 全球细胞和基因疗法的商业化。

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.