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

Area of Science:

• Biotechnology
• Bioprocess Engineering
• Separation Science

Background:

• Aqueous two-phase systems (ATPS) provide a scalable and cost-effective method for virus purification.
• Traditional batch ATPS methods face challenges in continuous operation and impurity removal.
• Optimizing continuous ATPS is crucial for efficient viral vector production.

Purpose of the Study:

• To develop and evaluate a continuous aqueous two-phase system (ATPS) for porcine parvovirus (PPV) purification.
• To assess the efficiency of continuous ATPS in removing protein and DNA contaminants.
• To explore methods for improving mixing, settling, and impurity removal in continuous ATPS.

Main Methods:

• A two-step ATPS using polyethylene glycol (PEG) and sodium citrate was adapted for continuous operation.
• Turbidity measurements (absorbance at 880 nm) were used to monitor mixing and settling efficiency.
• A flow-through anion exchange (AEX) filter was integrated for enhanced impurity removal.

Main Results:

• The continuous ATPS consistently achieved low DNA titers (<10 ng/mL) and recovered 66% of infectious PPV.
• Continuous operation resulted in higher protein contamination compared to batch processing, attributed to mixing/settling inefficiencies.
• Optimizing settling line material (PTFE) reduced settling time, and the AEX filter removed an additional 87% of host cell DNA without affecting protein removal.

Conclusions:

• Continuous ATPS is a viable method for scalable and cost-effective virus purification.
• Monitoring turbidity and optimizing system components can enhance the efficiency of continuous ATPS.
• Integration of sensors and filters can lead to automated, continuous purification processes for viral vectors.