Purification of a non-enveloped virus using sequential aqueous two-phase extraction.

Virus-based vaccines and therapies require a purification method that is both cost-effective and easily scalable. An aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and citrate salt has been proven to deliver high virus recoveries along with high impurity removal. However, these systems often place the virus into a viscous PEG-rich phase or at the two-phase interface, leading to difficulties in subsequent downstream processes. This study explored a second ATPS to extract the virus product back into the citrate-rich phase by changing the chemical conditions, a required step for future application of ATPS in industrial processes. ATPS performance was tested as a function of phase component concentration, phase component volume ratios, PEG molecular weight, salt type, pH, and glycine addition to identify the most impactful parameters for the extraction of non-enveloped porcine parvovirus (PPV). By shifting the pH, lowering phase component concentrations, and increasing the volume ratio of the citrate-rich phase between the first and second ATPS steps, 66 % of infectious PPV was recovered with 2.0 logs of host cell protein removal and 1.0 logs of host cell DNA removal. Using a PEG molecular weight of 8 kDa enabled a pH shift between the first and second ATPS steps without precipitation. Glycine addition during the first step of ATPS and phosphate salt use during the second step of ATPS did not significantly increase the overall recovery. In future studies, the optimized process will be implemented for multiple viral vector types and continuously to demonstrate continuous and low-cost viral vector manufacturing.