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Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography.

George PamenterLee DaviesCarol KnevelmanJames MiskinKyriacos MitrophanousDuygu DikiciogluDaniel G Bracewell
Published in: Biotechnology and bioengineering (2023)
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.
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