Enteroviruses are a genus of small, non-enveloped RNA viruses belonging to the family Picornaviridae and contain numerous clinically relevant serotypes. In particular, serotypes of enterovirus C have been shown to demonstrate oncolytic activity on cells overexpressing tumor-specific surface receptors and are currently in clinical trials as oncolytic viral immunotherapies. The purification of such live viral products can pose processing challenges such as labor intensive unit operations, low yields, and poorly scalable technologies such as gradient ultracentrifugation. These compound the interest in deploying chromatography-based purification processes for such products. While affinity-based steps can offer high selectivity, excellent impurity clearance, and significant volume reduction in an easily scalable unit operation, such applications are, with a few exceptions, rare within the area of viral products. A novel affinity chromatography capture step utilizing immobilized reduced glutathione (GSH) was recently developed for the direct capture of Coxsackievirus A21 produced from microcarrier cell-culture with high specificity and yields. Here, we demonstrate the wide applicability of GSH-affinity chromatography for the purification of multiple enterovirus serotypes. Viral stocks of Echovirus 1, Coxsackievirus A11, Coxsackievirus A13, Human Rhinovirus 1B, and Human Rhinovirus 35 were used to infect flasks containing static mammalian cell cultures. At the end of the virus production stage, viral fluid was harvested and purified using small scale high throughput chromatography columns packed with Glutathione Sepharose 4FF resin. Analysis of the elution products demonstrated that GSH affinity chromatography step maintained its mode of action for all tested enterovirus stereotypes and delivered concentrated and purified viruses.

Demonstration of a glutathione affinity chromatography capture step for multiple enterovirus serotypes

ACS Spring 2022

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