(239d) Characterization of Protein-Polymer Conjugates Using Aqueous Two-Phase Systems

Covalent attachment of polymers to proteins to produce bioconjugates with improved properties such as increased stability to external stimuli and longer circulation times in vivo has been accomplished using various methods over the last few decades. Traditionally, a linear or branched polyethylene glycol chain is attached to a protein to give the conjugate these improved properties. Many different polymers, polymer structures, and combinations of polymers may also be used to give the conjugate not only improved stability but also specific functionality. As more complex protein-polymer bioconjugates are developed and utilized therapeutically and industrially, there is an increasing need to characterize and purify the conjugated products of the reaction. This characterization may help spur future developments in the areas of bio-therapeutics, biosensors, biotechnology, and enzyme ruggedization among others. Extraction using aqueous two-phase systems presents an opportunity to characterize conjugates in a mild environment, retaining conjugate function and allowing future use of the conjugate.

In this work, aqueous two-phase systems were evaluated for the separation of protein-polymer bioconjugates on both benchtop and millifluidic scales. Protein-polymer conjugates were formed by atom transfer radical polymerization from initiation sites attached to the protein. This polymerization method allows for the creation of polymers with different structures in aqueous conditions and leads to simple purification of the conjugate from the reaction mixture. A millifluidic device was used to form two-phase droplets of controlled composition surrounded by an immiscible carrier fluid for quicker analysis and less material usage than on the benchtop scale. This high-throughput method allowed many conditions to be tested while minimizing the number of conjugate reactions required. Selected benchtop experiments were performed to validate the millifluidic approach. By varying the phase-forming compositions and components, the protein to which polymers were attached, and the attached polymer identity and length, a series of partition coefficient data was obtained. The results suggest that protein-polymer conjugates partition much differently than the native protein at the same conditions. Partitioning of high polymer grafting density conjugates is compared to the partitioning of an equivalent amount of polymer in an aqueous two-phase system and the results are presented here. We also consider multi-step extraction to purify conjugates with similar physical structure.