Tie line framework to optimize non-enveloped virus recovery in aqueous two-phase systems

Area of Science:

• Biotechnology
• Downstream Processing
• Virology

Background:

• Viral particle purification is complex, requiring methods that maintain virus activity while removing contaminants.
• Current purification methods are often expensive and inefficient.
• Aqueous two-phase systems (ATPSs) present a promising alternative for cost-effective viral purification.

Purpose of the Study:

• To investigate the purification efficiency of ATPS for non-enveloped viruses, specifically porcine parvovirus (PPV) and human rhinovirus (HRV).
• To evaluate the impact of thermodynamic tie-line length (TLL) on virus partitioning and recovery in a polyethylene glycol (PEG)-citrate system.
• To understand the role of hydrophobic and electrostatic forces in virus purification using ATPS.

Main Methods:

• Utilized a polyethylene glycol (PEG) 12 kDa-citrate ATPS at pH 7.
• Investigated virus partitioning across three different thermodynamic tie-line lengths (TLLs).
• Assessed recovery of infectious PPV and partitioning of HRV, alongside contaminant removal (proteins and DNA).

Main Results:

• Increasing TLL in the PEG-citrate system significantly enhanced virus recovery in the PEG-rich phase.
• Maximum recovery of 79% for infectious PPV was achieved at a TLL of 36 w/w% and a tie-line ratio of 0.1.
• Contaminants (proteins and DNA) were primarily partitioned into the citrate-rich phase, demonstrating effective purification.

Conclusions:

• ATPS, particularly the PEG-citrate system, is effective for purifying non-enveloped viruses like PPV and HRV.
• Virus recovery is dependent on the TLL, with longer TLLs promoting higher recovery due to increased hydrophobic and electrostatic driving forces.
• The differential partitioning is governed by surface hydrophobicity and charge, highlighting ATPS as a tunable platform for viral purification.