(374f) Determination of Binodal Curves and Tie Lines for Aqueous Two-Phase Systems with Osmolytes for Bioseparations

Aqueous two-phase system (ATPS) is considered a potential alternative to currently used expensive methods in the biopharmaceutical industry such as chromatography and filtration. ATPS, formed by two incompatible aqueous solutions, provide inexpensive, biocompatible, mild, and environment-friendly advantages over the conventional methods, and can be easily adapted to continuous processing. However, the lack of understanding of the driving mechanisms of biomolecules poses a major challenge for industrial implementation. Our recent investigation for the recovery of porcine parvovirus (PPV) in a PEG 12kDa-citrate system indicates that hydrophobicity of the viral particles and the system play a vital role in the virus partitioning. The partitioning was affected by the salting-out effect from the citrate-rich phase and hydrophobic interaction from the PEG-rich phase.To build on the hydrophobicity theory, we incorporated osmolytes in the system. Osmolytes are naturally occuring compounds that have a high affinity for water. Common osmolytes include sugars and amino acids. Glycine has shown to preferentially flocculate relatively hydrophobic viruses in a complex lysis mixture. Using this property of osmolytes, we hypothesized that osmolytes would increase the hydrophobic driving force in ATPS. The driving force can be examined by generating the binodal curves and tie lines. The binodal curve separates the homogeneous region from the two-phase region and the tie line represents the systems separating into two-phases having the same PEG-rich and citrate-rich phase compositions. We have determined the binodal curves for PEG 12kDa-citrate by including 0.5M glycine or betaine (trimethyl glycine) at pH 7. The binodal curves shifted towards the origin as compared to the system without osmolytes, suggesting an elevated driving force to form two phases.Tie lines were determined by conductivity measurements of the citrate-rich phase. The osmolyte concentrations were determined in the citrate-rich phase by LC-MS. The tie line slope of the glycine system was found to be greater than the betaine system. This demonstrated that any two-phase system formed with glycine will have higher concentration of citrate and lower concentration of PEG in their respective phases as compared to betaine. The osmolytes were found to be in the citrate-rich phase. The occurrence of the osmolytes in the citrate-rich phase will help in salting-out the virus from the citrate-rich phase, providing more separation driving force within the ATPS.