Non-infectious particle removal in lentiviral vector purification: A path to higher quality

Lentiviral vectors (LV) are the preferred gene therapy tools when sustained, long-term transgene expression is required. However, their clinical application is hindered by high manufacturing costs and complex ex vivo protocols. Unlike adeno-associated viruses (AAV), impurities in LV productions remain poorly characterized. This knowledge gap hampers the development of more efficient purification strategies and safer therapies suitable for in vivo administration. In this study, particles in HEK293T-derived LV feedstocks were characterized using advanced analytics, showcasing a small percentage of intact LV (∼6%), and an overwhelming majority (∼94%) consisted of non-functional impurities such as VSV-G empty particles and extracellular vesicles (EV). To address this, a negative-mode affinity chromatography process was developed to selectively remove impurities while retaining functional LV. Initially, the selectivity of several V H H binders for closely resembling impurities and their lack of interaction with functional vectors was assessed. Spin columns packed with functionalized resins against tetraspanins CD81, CD63 and CD9 were evaluated on phenotyped LV feedstocks (ΔVSV-G, Δp24, and ΔN-glycans) generated via modified transfections. Standard LV and non-specific binders served as controls. Binder performance was evaluated using ELISAs (p24, VSV-G, CD63, CD81, CD9), digital droplet PCR, and infectivity assays. Among the binders, anti-CD63 emerged as the leading candidate, preserving 87 ± 6% of infectious, full-genome LV while removing 87 ± 1% of chimeric, non-infectious particles sharing LV components. This novel negative-mode affinity approach for impurity removal represents a significant advance for LV manufacturing. Enhanced purity and product quality may enable reduced therapeutic doses and improved clinical outcomes, paving the way for next-generation LV purification strategies.