Isolere Bio Inc

Lentitag: a Novel Approach to the Efficient Manufacturing of Active Lentiviral Vectors - 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.