Issue link: https://www.e-digitaleditions.com/i/1248960
Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING 2020 25 One challenge presented by lentiviral vectors is that they are relatively large biomolecules and are more challenging to process than monoclonal anti- bodies (mAbs). Conversely, as a result of "budding" out of the host cell, lentiviral vectors have a viral lipid envelope covered with portions of the host cell membranes. This results in a net charge, which makes the lentiviral vector more likely to adhere to processing materials, including membranes. As such, process parameters must be carefully con- trolled to avoid and/or reduce shear stress and op- timize virus yields. This article describes the results of a collabora- tion which focused on identifying best practices during clarification of the bulk harvest to ensure maximum virus vector yield. Considerations for clarification of lentiviral vectors Most modern depth filter technologies were devel- oped for use with mAbs, the majority of which are manufactured in Chinese hamster ovary (CHO) cells. Similar to CHO cells in which antibodies are transported outside the cells, lentivirus particles are also transported to the extracellular space, eliminat- ing the need for a lysis step. This dramatically re- duces host cell protein (HCP), nucleic acid, and cell debris contaminants that would typically need to be removed via the downstream process. Having cells remain intact also reduces particle size distribution with a bias towards the high end, which increases capacity, reduces breakthrough of filtration opera- tions, and increases centrifugation efficiency. While CHO cells can be slightly larger than HEK-239T cells, both behave in a similar fashion, which enables the use of existing market clarifi- cation technology, with some notable caveats as described below. There are four main clarification technologies routinely implemented in various combinations for mammalian cell operations—centrifugation, tan- gential flow filtration, depth filtration, and normal f low membrane filtration. Centrifugation. Continuous disc stack centrifuga- tion has been the mainstay of bioprocessing clari- fication; centrifuges are highly economical and Figure 1. Typical lentiviral vector production process. Thaw cells Seed train Cell expansion in bioreactor Plasmid transfection Virus production Clarification Nuclease Treatment Purification with Chromatography (1-2 steps) Diafiltration with Tangential Flow Filtration (TFF) Final filtration Fill in assemblies Storage Diafiltration with Tangential Flow Filtration (TFF) ALL FIGURES COURTESY OF THE AUTHORS.