Background: Modified Vaccinia Ankara (MVA) vectors are highly immunogenic vaccine platforms for the delivery of recombinant antigens. Efficient downstream processing is still challenging, particularly because substantial fractions of the virus remain intracellular. While chemical cell lysis that releases MVA particles into the supernatant before clarification can greatly enhance process efficiency and scalability, this step remains insufficiently characterized. Methods: This study assessed the compatibility of ionic, non-ionic, and zwitterionic detergents with the virus as purification target. Polysorbate 20 (Tween 20) was selected as a candidate detergent and evaluated across harvest times of 48–72 h post-infection (hpi) at concentrations of 0.01–0.5% (v/v). Results: The addition of 0.01% to 0.05% Tween 20 at 48 hpi resulted in a twofold increase in supernatant virus within one hour of application. Extended exposure to Tween 20, combined with a 650 mM mixture of NaCl, NaBr, and KCl, promoted virus particle release. However, Tween 20 concentrations above 0.1% reduced MVA infectivity. A filtration cascade using pore sizes of 5 µm and 1.2 µm achieved product yields of 77–83% at 48 hpi and 41–69% at 72 hpi, respectively. Host-cell DNA is an important contaminant during viral vector processing. However, the application of 0.05% (v/v) Tween 20 resulted in a 35% reduction of dsDNA released into the culture supernatant; the nuclei could not be preserved intact under high-salt conditions to avoid the release of cellular DNA. Conclusions: In summary, this comprehensive data demonstrated that non-ionic detergents can be used to induce cell lysis while maintaining infectious activity of enveloped MVA.

Modified Vaccinia virus Ankara (MVA) is a potent vector for the development of vaccines and novel cancer treatment strategies. Since it is a non‐replicating virus in human recipients, high infective titers are required for therapeutic efficacy. Harvesting and clarification of cell culture‐derived MVA are critical for the overall process yield and remains challenging, as full MVA release depends on cell lysis, resulting in a difficult‐to‐filter cell lysate. In this study, we present the development of a high‐yield clarification strategy using diatomaceous earth (DE) as a natural filter aid, followed by an optimized endonuclease treatment. An ionic strength containing a 650 mM salt mixture of NaCl, NaBr, and KCl allowed impurity depletion by DE binding while maintaining a complete virus recovery. The optimal alluvial filtration conditions were achieved with a PURAFIX® CH 09 depth filter with a 10–30 µm retention rate. The optimal conditions were identified as a combination of a Celpure® C1000 precoat and a Celpure® C300 additive added to the feed. This robust process achieved 94% total virus recovery while reducing turbidity to 23% and residual DNA and protein impurities to 76% and 72%, respectively, in a single‐step filtration. Subsequent nuclease digestion benefits from a reduced particle and impurity burden, resulting in further 93% DNA depletion under optimal conditions of 11 h of incubation at 21°C with 51 U/mL of the salt active DENARASE® High Salt endonuclease. The study demonstrated that sterilized filter aids enable a single‐step filtration to recover high MVA titers with a reduced impurity burden, paving the way for improved downstream process efficiency.