(239f) Optimization Opportunities of Virus Purification in Different Modes of Aqueous Two-Phase System

Lack of platform technologies for downstream processing of viral based vaccines and gene therapy vectors have led to a boom in developing new technologies. Aqueous two-phase systems (ATPS) have been in the forefront of the unit operation candidates to provide a high throughput and high yield unit operation. ATPS, formed by two partially miscible aqueous solutions, are known for their inexpensive, mild, and environmentally friendly advantages. However, the discrepancy in understanding the partitioning mechanism of biomolecules is hindering industrial implementation. Using two non-enveloped viruses – porcine parvovirus and human rhinovirus, the partitioning mechanism in a polyethylene glycol (PEG)-citrate system was explored. The preferential partitioning of viruses to the PEG-rich phase was heavily influenced by the virus surface characteristics (surface charge and hydrophobicity), and relative phase characteristics (electrostatics and hydrophobicity). Furthermore, the opportunities in enhancing the driving forces by manipulating the system characteristics and compositions will be discussed. We have developed two separate methods to improve the driving forces: 1) osmolyte addition and 2) affinity peptide ligand addition. Osmolytes, naturally occurring compounds, changed the water structure in the system which resulted in 100% recovery of the viral particles in the PEG-rich phase at lower system compositions compared to systems without osmolytes. The other method involved a virus specific affinity peptide ligand conjugated to PEG. The affinity study performed with a peptide ligand specific to PPV showed a relative increase in the recovery as compared to the original systems. Overall, this talk will provide a scope to enhance the partitioning and guidelines to generalize the virus recovery process within a confined experimental space.