(239g) Using Droplet-Based Millifluidic Platform to Separate Biomolecules in Aqueous Two-Phase Systems

Aqueous two-phase systems (ATPS), formed by mixing two polymers or a polymer and a salt, has been used as a separation and purification tool for more than 50 years. With nearly 80% water content, ATPS can achieve separations with minimum damage to particles with delicate structures such as proteins. Because of its potential for continuous operation and process integration, ATPS is particularly promising to meet the downstream processing needs created by the fast-growing production rate of biomolecules. The partitioning behavior involved, however, is complex and difficult to predict due to the number of factors that can alter the process. The application of ATPS is therefore limited because of the inability to easily optimize the desired partitioning behavior in very large parameter space. Here, we propose using a droplet-based millifluidic platform to systematically investigate the impact of multiple factors on the partitioning behavior in an ATPS. The precise manipulation of micro-scale droplets allows for higher compositional resolution, shorter settling times, and lower sample consumption, making this technique an efficient and flexible tool for rapidly exploring a large parameter space. In millifluidic droplets, the partitioning behavior of hemoglobin in PEG-potassium phosphate salt two-phase system is characterized as a function of tie-line length, the PEG/salt concentrations, and the concentration of added salt. The amount of protein in each phase is inferred from absorbance monitored by an in situ spectrophotometer. The degree of partitioning can be manipulated so that more proteins go to the preferred phase. The condition for maximum partitioning is further explored to facilitate separation of two proteins in the same ATPS.