Continuous purification of a parvovirus using two aqueous two-phase extraction steps.

Aqueous two-phase systems (ATPS) are a liquid-liquid extraction method that offers low-cost, continuous-adaptable virus purification. A two-step ATPS using polyethylene glycol (PEG) and sodium citrate that recovered 66% of infectious porcine parvovirus with 2.0 logs of protein removal and 1.0 logs of DNA removal in batch has now been run continuously. The continuous system output of <10 ng/mL DNA regardless of starting DNA titer agreed with batch studies. However, the continuous system had a five-fold higher contaminating protein titer than batch studies, likely because of incomplete mixing or settling. Turbidity was tested as a measure of mixing and settling efficiency. Monitoring in-line absorbance at 880 nm directly after mixing and before collection in the settling reservoir could track both mixing and settling during operation. Settling time was reduced by changing the settling line material from PVC to PTFE, which is more hydrophobic. A flow-through AEX filter tested to make impurity removal more robust recovered 90% of PPV and removed an additional 87% of host cell DNA. The filter did not add any additional protein removal. In the future, in-line absorbance sensors could be implemented along with conductivity sensors to measure salt concentration, refractive index sensors to track the PEG-citrate interface, and scales to track mixer and reservoir volumes to enable automated, continuous ATPS. Our vision is to integrate continuous ATPS into a fully continuous end-to-end production for viral vectors.