Location:
Classroom 3, Sanderson Building, King's Buildings, University of Edinburgh
Date:
Time-Dependent Sorption Behaviour of Viral Vectors
Abstract
Viral vector processes are still maturing. Here we study two viral vectors: adenovirus (AV) and lentivirus (LV). The labile nature of these products is a concern when using adsorption-based separation. Downstream processing sequences involving UF/DF and anion exchange-based (AIEX) chromatography were investigated.
Despite AIEX’s extensive use for LV recovery, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of lentiviral vector adsorption and other types of vector delivery systems. Here we show 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. Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterising the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak.
In the analogous recovery of AV, nanofiber-based ion exchange chromatography was investigated at differing ligand densities. Yield and resolution was highly sensitive to the ion exchange ligand density and time adsorbed to the solid phase. Increasing Q amine ligand density improved the separation of intact AV capsids from host cell protein impurities and product-related impurities including free hexon (a major capsid coat protein) and replication-defective AV capsids that contained DNA but was also associated with poor recovery.
In summary for both vectors studied in this work minimising the time spent in the adsorbed state was shown to be a critical parameter in retaining high yield.
Speaker
Daniel G. Bracewell is Professor of Bioprocess Analysis at the UCL Department of Biochemical Engineering. He has made major contributions to fundamentally understanding the recovery of biological products. Generating over £10 million in research funds including collaborations with Thailand, India and the USA. He has authored more than 100 peer reviewed journal articles in the area to date and currently supervises 15 doctoral and postdoctoral projects, many of these studies are in collaboration with industry. One such project was the basis from which the spinout Puridify was created. It was acquired by GE Healthcare (now Cytiva) in 2017.
