The 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 %.

对针对遗传和获得性疾病的安全有效的基于病毒载体的疫苗和基因疗法的巨大需求加速了病毒载体的发展。一个突出的例子是 Orf 病毒，与其他病毒载体相比，它具有广泛的应用、卓越的功效和出色的安全性以及更低的致病性。然而，除了这些有利的属性外，还需要开发一种高效且可扩展的下游工艺。最近，我们筛选了用于 Orf 病毒纯化的潜在色谱固定相。基于这些先前的成就，我们为细胞培养衍生的 Orf 病毒开发了一个完整的下游工艺。所述方法包括基于膜的澄清步骤、核酸酶处理、空间排阻色谱、以及使用 Capto® Core 700 树脂的二级色谱纯化步骤。显示了该过程对多种不同 Orf 病毒载体的适用性，测试了两种不同的基因型。这些研究提供了对两种基因型应用开发的下游方案的可能性，并导致总病毒产量约为 64%，澄清的步骤回收率 >70%，色谱序列的回收率 >90%。最终产品的蛋白质浓度低于检测限，每 1E + 06 个感染性病毒单位约 1 ng 的最终 DNA 浓度类似于总 DNA 耗竭的 96-98%。这些研究提供了对两种基因型应用开发的下游方案的可能性，并导致总病毒产量约为 64%，澄清的步骤回收率 >70%，色谱序列的回收率 >90%。最终产品的蛋白质浓度低于检测限，每 1E + 06 个感染性病毒单位约 1 ng 的最终 DNA 浓度类似于总 DNA 耗竭的 96-98%。这些研究提供了对两种基因型应用开发的下游方案的可能性，并导致总病毒产量约为 64%，澄清的步骤回收率 >70%，色谱序列的回收率 >90%。最终产品的蛋白质浓度低于检测限，每 1E + 06 个感染性病毒单位约 1 ng 的最终 DNA 浓度类似于总 DNA 耗竭的 96-98%。