Isolation of Proteins and Extracellular Vesicles via Capillary-Channeled Polymer Fiber Stationary Phases
Date of Award
Master of Science (MS)
Dr. R. Kenneth Marcus
Dr. Terri F. Bruce
Dr. George Chumanov
Dr. Carlos D. Garcia
Capillary-channeled polymer (C-CP) fiber stationary phases have been developed by the Marcus lab as an alternative approach to traditional, commercially-available silica-based supports for biomacromolecule separations. C-CP columns are created with natural and synthetic base polymers in a variety of chemistries and shapes, allowing for customization of separations, while providing low material costs and ease of construction. The stationary phase is virtually nonporous with respect to the size of proteins and can operate under high linear velocities (~100 mm s-1) while maintaining relatively low backpressures. As a result of these hydrodynamic benefits, low flow resistance at high volume flow rates, and efficient mass transfer characteristics are realized making these columns attractive for biomacromolecule separations.
C-CP fiber phases were evaluated for their performance for intact protein separations under RP-HPLC conditions. The separation quality, operational characteristics, and protein dynamic loading capacity on the fiber phases are compared to commercially-available superficially porous and monolithic columns, which are tailored to protein separations. A trilobal (y-shaped) polypropylene fiber phase was employed to separate a synthetic mixture of five proteins (having diverse chemistries and molecular weights). The separation quality was evaluated based on the resolution, peak heights/recoveries, peak widths, and peak areas. The present work illustrates the unique ability to operate with better hydrodynamic characteristics while yielding comparable chromatographic performance and binding capacities to the commercial columns.
Recently, Marcus and coworkers have successfully extended the fiber stationary phases for the isolation and quantification of extracellular vesicles (EVs). EVs are 30-1000 nm membranous vesicles secreted from all cells that play pertinent roles in many biological processes such as targeting and transporting biomolecules to specific cells, engaging in cell-signaling, maintaining homeostasis, and regulating biological functions. Currently, traditional methods developed for the isolation of small quantities of EVs are plagued by limitations in terms of product purity, low recoveries, and very low throughput. To overcome the practical limitation of low throughput, polyester (PET) C-CP fiber-phases have been examined via a frontal loading and recovery analysis to determine the load, throughput, and recovery from various complex matrices.
Billotto, Lacey, "Isolation of Proteins and Extracellular Vesicles via Capillary-Channeled Polymer Fiber Stationary Phases" (2022). All Theses. 3838.
Author ORCID Identifier