Enhanced lentiviral vector recovery and separation using arginine hydrochloride with CIM QA monoliths

Lentiviral vectors (LVVs) offer distinct advantages including large payload capacity and stable transduction of non-dividing cells, making them well-suited for ex vivo modification of stem or immune cells. However, chromatographic purification of LVVs is hindered by low recoveries, co-elution of product related impurities, and vector instability. In this study, we evaluated arginine hydrochloride (ArgHCl) as an alternative eluent to sodium chloride using CIMmultus™ QA (CIM QA) monoliths. Screening of solution conditions identified that phosphate buffer concentrations between 100-200 mM enhanced infective particle stability. During anion exchange screening experiments using a CIM QA 96-well plate, ArgHCl improved both infectious particle and p24 recoveries, which was corroborated in linear gradient elution (LGE) experiments using the monolith in a disk format. Furthermore, fractions eluted with ArgHCl exhibited improved colloidal stability compared to those eluted with NaCl. Increasing the ArgHCl gradient endpoint concentration to 1.5 M ArgHCl yielded infectious particle recoveries of 71%. Analysis of gradient fractions with nanoflow cytometry revealed a two-peak elution profile, with the more strongly retained peak enriched in vesicular stomatitis virus glycoprotein (VSV-G) positive particles, corresponding to greater infectious particle recoveries. ArgHCl also improved the chromatographic resolution between the initial impurity peak and the secondary peak, enabling improved separation. These findings support the use of ArgHCl and phosphate buffers to enhance recovery during the purification of LVVs using AEX chromatography and highlight the utility of nanoflow cytometry for rapid detection of product-related impurities.