Efficient Cell Removal and Impurity Reduction for Poxvirus Purification in a Single Step Alluvial Filtration.

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.