NSF Award
2404088
With support from the Chemical Measurement and Imaging Program in the Division of Chemistry and the Established Program to Stimulate Competitive Research (EPSCoR), Professor Ken Marcus and his research group at Clemson University will characterize and implement a unique format of fiber, capillary-channeled polymers (C-CP), as stationary phases for the isolation, quantification and characterization of biological nanoparticles (BNPs). Initial demonstrations of nanoparticle purification involved exosomes, a class of extracellular vesicles (EVs) from diverse biological media, via a hydrophobic interaction chromatography (HIC) protocol on polyester C-CP fiber columns. EVs are integral components in intracellular communication, and thus are involved in the evolution of many disease types. The same analytical approach has been extended to the isolation of lentivirus particles and adeno-associated virus (AAV) particles. These three classes of BNPs are similar in many physical and chemical aspects, which tend to make them very difficult to isolation by most chromatographic methods. Separations on C-CP phases represent a completely different paradigm to methods currently employed in the processing of BNPs. Approaches developed would touch aspects of fundamental biochemistry, clinical diagnostics, and gene therapy delivery; the latter being an area of intense interest and importance. The goal of the studies is to produce efficient and selective separations in an economical manner. This work is interdisciplinary, involving collaborative efforts between research groups in chemistry, bioengineering, and biological sciences. The project includes investigators affiliated with the Clemson University Center for Advanced Engineering of Fibers and Films. If successful, the project may have long term implications for the textile and biopharmaceutical industries. <br/><br/>The proposed studies are directed at exploiting C-CP fiber platforms to affect highly productive/ selective separations in the realm of bionanoparticle (BNP) analytics. The vast majority of separation/purification strategies for these species involve physical means such as ultracentrifugation, ultrafiltration, and size-exclusion methods. Use of C-CP fibers to affect these separations rests on the ability to differentiate BNPs based upon chemical functionality, whose interaction strengths are determined in-part based on particle size and the solvents employed to effect the separation. The C-CP fiber platform can be implemented across a number of formats including spin-down micropipette tips and microbore chromatography columns, as such, relevant problems in fundamental biochemistry, clinical analysis, and process analytical chemistry can be addressed. In-line absorbance detection for quantification, followed by particle sizing/counting via multi-angle light scattering (MALS), provides greater information about eluted nanoparticles/vesicles. This approach will be complemented by the use of novel instrumentation which provides particle sizing and number density via light scattering, but also specific BNP identification through fluorescence immunoassays, here for discrete samples. Multidimensional separations of target exosomes from cell cultures typically used to produce monoclonal antibodies (mAbs) brings a completely novel aspect to potentially achieve greater value from a single fermentation operation. In all cases, performance metrics will be directly compared to commercial columns and standard methods employed for nanoparticle isolation/purification. Development of practical methods will be augmented with fundamental studies directed at understanding physico-chemical processes at the microscopic level.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
