(235g) Development of Tie Lines for Optimization of Porcine Parvovirus Recovery in Aqueous Two-Phase System  

Improvements are needed in the downstream processing of viral vaccines, due to the increased complexity of the upstream processing, elevated demand, and the urge to reduce production costs. To escalate the purification process throughput, there is a demand for a shift from batch processes to continuous. Aqueous two—phase systems (ATPS) provides an inexpensive, biocompatible, and environmentally friendly method to purify viruses that can easily be adapted to continuous processing. Recent progress in increasing ATPS recovery has created a space for ATPS to become a potential substitute for more expensive unit operations, like density gradient centrifugation and chromatography. The main drawback that is holding back commercial production from using ATPS is the poor understanding of the partition mechanism of biomolecules. We are taking a thermodynamic approach to ATPS by studying the separation of virus particles along the tie-lines that connect the binodal two-phase region. Moving along the tie-lines changes the volume of each phase while keeping the concentration of the PEG and citrate in each phase constant. This shift in volume of the phase is hypothesized to change the purification and concentration of the viral product. The common system of aqueous polymer—salt was used to optimize the virus recovery. Porcine parvovirus (PPV), a 20 nm diameter, non-enveloped, single-stranded-DNA virus, was used as a model virus. We created the binodal curves for the PEG 12000-citrate salt system at pH 7 using the turbidimetric method and the tie lines were determined by measuring the concentration of citrate in each phase.  Infectious virus titer was used to determine the partitioning of the virus to the different phases. To this date we have checked different PEG-citrate configurations on the tie line length (TLL) of 36%w/w. We obtained a similar infectivity of ~log 5 MTT₅₀/ml of the PEG-rich phase by shifting the system on the selected TLL but the increasing trend for the yield was seen with the increasing volume of the PEG-rich phase as we move down the tie line. The citrate-rich phase contained minimum infectivity and the main shift was the amount of virus that was sequestered at the interface. We are looking for the same trend with different TLL and obtain a trend to optimize the recovery process. This approach to optimize the recovery/purification of PPV will aid in faster process development and will help to develop the key process parameters that effect ATPS separation of viral particles.