Defective interfering particles (DIPs) of Influenza A virus (IAV) are under investigation as broad-spectrum antivirals because they can strongly inhibit virus replication in vivo. An engineered OP7 chimera DIP was previously created and shown to have substantially improved antiviral efficacy compared with conventional IAV DIPs. High-yield production of OP7 chimera DIPs was earlier demonstrated in cell culture using perfusion mode. However, perfusion processes can be technically complex to implement at many production sites.
The principal aim of this work was to develop a fed-batch production process for OP7 chimera DIPs that retains advantages of high productivity while offering simpler integration into existing manufacturing facilities. The authors sought a feeding strategy that would overcome the common cell density effect and enable productive infection and DIP accumulation at high cell densities in stirred-tank reactors. Demonstrating robust seed virus trains and availability of GMP-compatible cell and virus banks for later biomanufacturing were additional goals.
The authors established a fed-batch cell culture process using a feeding regimen specifically designed to bypass the cell density effect that often limits virus production at high cell concentrations. Stirred-tank reactors were used as the production platform, and the process parameters were adjusted to enable productive OP7 chimera DIP infection up to high viable cell densities. Details on media composition, feed schedule, or exact process parameters were not reported in the abstract and thus are not available from the source text.
Using the implemented feeding strategy, productive virus infection was maintained in stirred-tank reactors up to a cell density of 10E+6 cells/mL. The fed-batch process produced a high virus titer measured at 3.8 log10(HAU/100 µL). This titer corresponds to a 3.4-fold increase in virus yield relative to a reference batch production process reported by the authors.
Material generated in the fed-batch process demonstrated a significantly increased interfering bioactivity in an in vitro potency assay compared with the reference batch material. The fed-batch–produced OP7 chimera DIPs therefore not only reached higher measurable titers than batch but also showed improved functional antiviral activity in the applied in vitro test.
Although the fed-batch OP7 chimera material exhibited enhanced potency and higher yields than the reference batch, virus titers were approximately twofold lower than those obtained previously using perfusion production of OP7 chimera DIPs. Importantly, despite the lower titers, the antiviral efficacy of the fed-batch material was reported to be similarly high as that of the perfusion-produced material when assessed by the in vitro potency assay.
The authors report demonstration of robustness of OP7 chimera DIP seed virus trains, an important attribute for reproducible manufacturing. Furthermore, corresponding GMP MDCK-PB2(sus) cell banks and OP7 chimera virus banks are now available for subsequent biomanufacturing efforts. These resources represent preparatory steps toward pharmaceutical-grade production under Good Manufacturing Practice (GMP) conditions.
The established fed-batch process offers a more straightforward, less technically complex alternative to perfusion for manufacturing OP7 chimera DIPs while maintaining high antiviral potency. Achieving productive infection up to 10E+6 cells/mL and a titer of 3.8 log10(HAU/100 µL) with a 3.4-fold improvement over batch supports the fed-batch route as a viable option for GMP manufacturing and later scale-up. The availability of GMP-compatible cell and virus banks and demonstrated seed-train robustness further position this process for translation to clinical-grade production and larger-scale manufacturing.
A patent for the use of OP7 as an antiviral agent to treat IAV infection is approved in the USA and pending in the European Union and Japan; patent holders are S.Y.K. and U.R. A further patent for OP7 use against coronavirus infection is pending with the same patent holders. Besides these patent disclosures, the authors declare no other conflicts of interest. Funding declared in the source was from Max Planck Innovation, MTTADYSY006.