Highlights Estimation of the isoelectric point and size of Vero cell-derived Orf virus. Limited dynamic binding capacity of tested Orf virus to sulfated cellulose. Purification of Orf virus by steric exclusion chromatography lead to 84 % recovery. Hydrophobic interaction chromatography suitable for Orf virus purification. Promising unit operations for a scalable DSP to produce Orf virus viral vectors.

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Steric exclusion chromatography (SXC) is a promising purification method for biological macromolecules such as the Orf virus (ORFV) vector. The method’s principle is closely related to conventional polyethylene glycol (PEG) precipitation, repeatedly implementing membranes as porous chromatographic media. In the past decade, several purification tasks with SXC showed exceptionally high yields and a high impurity removal. However, the effect of varying process parameters, on the precipitation success and its limitations to SXC, is not yet well understood. For this reason, the precipitation behavior and SXC adaptation for ORFV were investigated for the PEG/ORFV contact time, the membranes pore size, and the type and concentration of ions. All three parameters influenced the ORFV recoveries significantly. A small pore size and a long contact time induced filtration effects and inhibited a full virus recovery. The application of salts had complex concentration-dependent effects on precipitation and SXC yields, and ranged from a complete prevention of precipitation in the presence of kosmotropic substances to increased efficiencies with Mg2+ ions. The latter finding might be useful to reduce PEG concentrations while maintaining high yields. With this knowledge, we hope to clarify several limitations of SXC operations and improve the tool-set for a successful process adaptation.

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