Two-stage chromatographic purification of lentiviral vectors with enhanced particle characterization via nanoflow cytometry

Abstract

August2025School of EngineeringLentiviral vectors (LVVs) are central to the advancement of gene therapy, particularly in ex-vivo applications such as chimeric antigen receptor T (CAR-T) cell therapy. However, existing purification methods of LVVs face significant challenges, including process variability, colloidal instability, and low functional recovery. This thesis addresses these limitations through the development of a two-stage, orthogonal chromatographic purification process designed to enhance both recovery and stability of LVVs.Anion exchange chromatography (AEX) remains a foundational unit operation in LVV purification, but the strong electrostatic interactions between LVVs and quaternary amine ligands often necessitates high salt concentrations for elution, which can compromise vector integrity. Additionally, conventional bead-based stationary phases rely on pore diffusion to utilize their total binding capacity- however, the size of LVV inhibits its diffusion into traditional bead-based media. To overcome these limitations, we evaluated monoliths, which enable large modalities like LVV to access the entire functionalized surface area via convective transport, resulting in improved mass transfer, higher binding capacity, and faster processing times, enabling greater recoveries. We evaluated Arginine hydrochloride (ArgHCl) for its potential to improve recovery and colloidal stability during the CIM QA step. Arginine is a widely used formulation additive known for enhancing protein solubility, suppressing aggregation, and improving elution profiles. ArgHCl substantially improved physical and infectious recovery in linear gradient elution experiments. In addition to improving recovery, dynamic light scattering measurements revealed that ArgHCl enhanced the colloidal stability of eluted fractions compared to the conventional NaCl eluent, where particle aggregation was evident. Nanoflow cytometry was then employed to characterize particle heterogeneity and assess particle retention behavior of VSVG-positive and VSVG-negative subpopulations across LGE experiments. This analysis revealed a 2-peak elution profile on CIM QA, with a primary peak consisting predominantly of VSVG-negative particles and a secondary peak enriched for VSVG-positive particles, which corresponded to infectious recovery. The tetraspanin protein CD9, a known exosomal marker protein, was found to be enriched in the initial impurity peak of LGE fractions. The impurity fraction containing the highest levels of CD9 was then recombined with infectious particles from the secondary peak, resulting in a measurable reduction in infectivity. These findings suggest that exosomes constitute a substantial portion of the initial impurity peak and may impair LVV transduction efficiency if not effectively removed during purification. Following AEX development, a secondary flowthrough polishing step using Capto Core 700 was implemented to further reduce host cell protein (HCP) and double-stranded DNA (dsDNA) impurities. Improved LVV recoveries were observed when low concentrations of ArgHCl (150–300 mM) were added in the feed, without compromising impurity clearance. As a result, the Capto Core step was positioned second to the CIM QA capture stage. When operated in tandem, the integrated two-step process achieved high recoveries of both vector transgene and p24, along with a 2-log reduction in HCP and dsDNA levels. Overall, this study establishes a two-stage purification process that enables effective impurity clearance and high LVV recovery.Ph