Time‐dependent sorption behavior of lentiviral vectors during anion‐exchange chromatography

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion‐exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV‐1‐LV recovery over quaternary‐amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order‐like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes (t Y 1 / 2 ${t}_{{Y}_{1/2}}$ = 12.7 and 18.7 min). Upon gradient elution, a two‐peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows.