Lothert, K.K.LothertPagallies, F.F.PagalliesEilts, F.F.EiltsSivanesapillai, A.A.SivanesapillaiHardt, M.M.HardtMoebus, A.A.MoebusFeger, T.T.FegerAmann, R.R.AmannWolff, M.W.M.W.Wolff2022-03-062022-03-062020https://publica.fraunhofer.de/handle/publica/26528610.1016/j.jbiotec.2020.08.014The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96-98 %.en540660571572journal article