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1 Introduction Viral vectors are modified viruses that deliver therapeutic genetic material to target cells and are an emerging platform for gene therapy ( A prototype strain of coxsackievirus A21 (CVA21), a non-enveloped enterovirus in the Picornaviridae family, is undergoing clinical evaluation as an oncolytic virus immunotherapy application for the treatment of several types of cancers ( Based on reports that enteroviruses interact with intracellular antioxidant glutathione (GSH) during capsid assembly in infected cells ( The objective of this work was to establish a robust and scalable purification method of CVA21 with high infectivity yield and purity that can accommodate variable cell culture conditions