Cell and gene therapies are garnering attention for their remarkable clinical outcomes and their promise to treatdiseases previously considered uncurable. Lentiviral vectors (LVs) are used in over one third of applications incell and gene therapies.2 As with most viral vector and vaccine manufacturing, LVs are challenging andexpensive to manufacture and there are no methods in commercial use that offer specificity, high yield, andscalability. Current manufacturing relies on anion exchange chromatography, which (1) has poor capacities forlarge viruses that cannot effectively diffuse into small resin pores, (2) necessitates additional purification stepsbecause its lack of LV specificity causes contaminant co-purification, and (3) requires harsh elution conditionsthat result in low infectious LV recoveries of only ~50%.15, 1 In this Phase I SBIR proposal, Isolere Bio will developa fusion protein reagent comprised of an affinity domain that is specific for LV and a proprietary biopolymerdomain that has robust and precisely tunable liquid-liquid phase separation behavior. The LentiTag⢠reagentwill capture LVs in solution with high specificity and then efficiently sequester them into phase separated dropletson command with a simple environmental trigger - an incremental adjustment in salt. These droplets, highlypure 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 othercellular contaminants have been washed away, the LV can be gently recovered from the droplets with an elutionbuffer that disrupts the affinity interaction at near-neutral pH. To demonstrate LentiTagâ¢'s technical feasibility,Isolere will first design, produce, and characterize an LV-specific capture protein (LCP). The LCP binds the mostcommon 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 performhigh-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 thestandard industry process, quantifying final LV concentration, infective titer, yield, purity, and total processingtime. Last, because the LCP is well-hydrated and sterically occluding in its soluble state, we also hypothesizethat it can confer protective and stabilizing effects to LVs, which are prone to progressive activity loss andaggregation during storage. Our final aim is to explore the impact of our reagent on LV stability, both duringupstream production (preventing host cell auto-transduction) and as an additive to formulated LV stored attemperatures ranging from -70ºC to 37 ºC. The LentiTag⢠technology will provide a scalable platform for LVmanufacturing that will help to both democratize and accelerate the discovery, development, andcommercialization of cell and gene therapies around the globe.
Public Health Relevance Statement: PROJECT NARRATIVE
Lentiviral vectors (LVs) have become increasingly utilized in cell and gene therapy applications, but their
commercial manufacturing remains challenging and costly because of the dearth of purification methods that are
efficient and scalable. Isolere Bio, Inc. is developing LentiTagâ¢, a novel phase separation technology that can
capture LVs with high specificity and then, on command, sequester the virus into pure, concentrated droplets
that can be separated from contaminants with simple, scalable filtration. LentiTag⢠will significantly improve the
10-25%1 recovery yield achieved with today's standard industry methods and it may also address the storage
and stability issues inherent to labile LVs, thereby making these important vectors more globally accessible.
Project Terms: | | | |
