Optimizing the clarification of industrial scale viral vector culture for gene therapy

Abstract

Viral-based vector systems such as lentivirus (LV) and adeno-associated virus (AAV) are widely used and show great potential for delivery of genetic material to target cells in gene therapy. Downstream processing of LV and AAV offers its own unique challenges to generate clinical products of high titer, high potency, and high purity. For AAV, downstream challenges include the undesired production of empty capsids, and the process typically requires a cell lysis step, which generates a significant amount of host cell contaminants. In the case of LV, downstream challenges include low virus stability due to the presence of a fragile lipid envelope layer, as well as sensitivity to pH variations, salt concentrations, and shear stress. The objective of this work was to identify an efficient clarification strategy to remove a wide range of impurities found in typical adherent and suspension based viral vector cell culture. These include host cells, cell debris, aggregates, and cell culture media components. The clarification step needs to combine high throughput for impurity removal, high product yield, and ease of scale-up to prepare for downstream operations. Our testing evaluated various depth filters, prefilters, and bioburden reduction membrane filters, made up of different organic and inorganic materials, to clarify the viral vector cell culture. Both AAV and LV processes can be adherent or suspension based, each having their own challenges. In this work, we compared clarification options in terms of throughput and recovery for adherent LV and suspension AAV viral vector feed streams.

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

“…In addition to affinity chromatography, ion exchange chromatography has been extensively explored as an initial capture step. Due to the net charge on the surface of the capsid at a wide range of pH (pI 5.9-6.3; Qu, Wang, Wu, & Xu, 2015), ion exchange chromatography can work as a capture step but often cannot be highly selective due to the number of charged impurities present in the capture load. This results in the need for multiple purification steps.…”

“…The packaged DNA is believed to interact with the surface proteins of the capsid and change the net charge present on the capsid surface. Based on literature, the mean calculated pI value of all the AAV's empty capsids is estimated to be 6.3 and full capsids estimated to be 5.9 (Qu et al, 2015), which enables this separation, although exact values will vary based on serotype. Impurities like HCP and DNA have both a large size and pI difference that also allows for separation using ion-exchange based chromatography (IEX) due to the difference in surface charge at given conditions.…”

“…Tangential flow filtration (TFF) is the standard method that is performed and utilizes either flat sheet filter cassettes or hollow fibers (Clément & Grieger, 2016). A poly ethyl sulfate (PES)-based membrane with a molecular weight cut-off (MWCO) of 100 kDa or 300 kDa is recommended as AAV has a molecular weight over 600 kDa (Qu et al, 2015), though it is typically preferred in standard TFF process design to have a MWCO three-fold lower than the molecule of interest. While PES or modified PES constitute a large percentage of the membranes used in the biopharmaceutical industry, manufacturers also utilize polysulfone, regenerated cellulose, or mixed cellulose ester.…”

Lentiviral Vector Bioprocessing

“…Filtration techniques, which are effective and scalable, dominate initial clarification, with disposable systems offering simplified cleaning and validation. The use of cascading filters, whereby the feed is filtered with progressively finer filters can prevent premature fouling of the end filter that may result in titre loss due to vector exclusion and extended process times due to reduced flux [ 182 , 183 ]. The choice of filter impacts the efficiency of the clarification step.…”

Evolution of culture systems for viral vector production: advantages, challenges and cost considerations

“…There are also implications for the early part of the downstream process; a recent study we performed has shown that different clarification strategies are required depending on whether the virus is manufactured in adherent or suspension format [1].…”