Abstract
Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.
Highlights
Infections with influenza A virus (IAV; list of abbreviations, Table 1) result worldwide in up to 650,000 deaths annually (Iuliano et al 2018)
The metabolite uptake rates of Madin-Darby canine kidney (MDCK)-PB2(sus) cells grown in perfusion cultivations were very comparable to those reported previously for batch cultivations and to data obtained for the parental suspension cell line (Hein et al 2021a)
When a perfusion process with the same medium but a different MDCK cell line was established, a lower cell-specific perfusion rate (CSPR) from 60 pL/ cell/day was sufficient (Wu et al 2021)
Summary
Infections with influenza A virus (IAV; list of abbreviations, Table 1) result worldwide in up to 650,000 deaths annually (Iuliano et al 2018). Methods to produce IAV DIPs focused on eggbased manufacturing with STV co-infection (Dimmock et al 2008) This resulted in a relatively high variability of the produced material (contamination with multiple other DIPs). Adherent Madin-Darby canine kidney (MDCK) cells were genetically modified to express the polymerase basic protein 2 (PB2) encoded by IAV segment 1 (Seg1) vRNA. All mice infected with an otherwise lethal dose of IAV survived the infection when treated with DI244 These experiments already clearly demonstrated the antiviral potential of these purely clonal DIPs. the DI244 material evaluated in this study was produced in shake flasks only and large DIP quantities were required. A tubular membrane called virus harvest unit (VHU, pore size ~ 10 μm) was used for continuous virus particle harvesting during the perfusion process
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