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rVSV-Spike (rVSV-S) is a recombinant viral vaccine candidate under development to control the COVID-19 pandemic and is currently in phase II clinical trials. rVSV-S induces neutralizing antibodies and protects against SARS-CoV-2 infection in animal models. Bringing rVSV-S to clinical trials required the development of a scalable downstream process for the production of rVSV-S that can meet regulatory guidelines. The objective of this study was the development of the first downstream unit operations for cell-culture-derived rVSV-S, namely, the removal of nucleic acid contamination, the clarification and concentration of viral harvested supernatant, and buffer exchange. Retaining the infectivity of the rVSV-S during the downstream process was challenged by the shear sensitivity of the enveloped rVSV-S and its membrane protruding spike protein. Through a series of screening experiments, we evaluated and established the required endonuclease treatment conditions, filter train composition, and hollow fiber-tangential flow filtration parameters to remove large particles, reduce the load of impurities, and concentrate and exchange the buffer while retaining rVSV-S infectivity. The combined effect of the first unit operations on viral recovery and the removal of critical impurities was examined during scale-up experiments. Overall, approximately 40% of viral recovery was obtained and the regulatory requirements of less than 10 ng host cell DNA per dose were met. However, while 86-97% of the host cell proteins were removed, the regulatory acceptable HCP levels were not achieved, requiring subsequent purification and polishing steps. The results we obtained during the scale-up experiments were similar to those obtained during the screening experiments, indicating the scalability of the process. The findings of this study set the foundation for the development of a complete downstream manufacturing process, requiring subsequent purification and polishing unit operations for clinical preparations of rVSV-S., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)