Generation of CAR-T cell therapies relies predominantly on lentiviral vectors (LVs). As in vivo CAR-T trials progress, demand for larger quantities of higher-potency LV is expected. However, primary capture by anion-exchange chromatography (AIEX) remains a major manufacturing bottleneck from use of adsorbents not tailored to LVs, leading to complex binding phenomena such as time-dependent product loss and a heterogenous two-peak elution profile arising from two distinct LV subpopulations (Peak 1 and Peak 2). Current industry practice is to collect both peaks to maximize total product recovery. Yet peak-specific differences in product potency and demand for higher purity products for use in in vivo CAR-T cell therapy will likely drive a need to separate these LV species. Electrospun nanofibers offer a promising next-generation adsorbent for addressing these challenges, making it essential to understand how Q-nanofiber design affects LV recovery and Peak 1/Peak 2 selectivity. This work indicates that Peak 1 and Peak 2 LVs differ in their T-cell transduction potency. Despite generating higher numbers of CD3+GFP+ T cells at low doses, Peak 1 sees reducing T-cell viability and %CD3+GFP+ cells at higher doses, likely from an inhibitory effect of VSV-G vesicles that are co-eluted with Peak 1 LV during AIEX. In contrast, Peak 2 LV maintained increasing %CD3+GFP+ T cells and high viability across doses. A Q-nanofiber adsorbent enabling high-recovery AIEX while increasing LV peak separation was therefore required to isolate the more potent Peak 2 LV subpopulation. Increasing nanofiber porosity (0.4-0.6 v/v) enhanced LV recovery by reducing time-dependent loss, whereas Peak Separation increased with ligand density and nanofiber diameter. An optimal Q-nanofiber structure was therefore identified and validated across two LV batches, achieving high recovery (64%-82%) while isolating Peak 2 LV from the major VSV-G elution.
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