Date of Award
5-2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
Committee Chair/Advisor
R. Kenneth Marcus, Committee Chair
Committee Member
Carlos Garcia
Committee Member
Brian Dominy
Committee Member
Sarah Harcum
Abstract
Capillary-channeled polymer (C-CP) fibers as an alternative support/stationary phase for biomacromolecule separations on analytical and preparative scales have been developed by Marcus research group. The combination of the fiber micro- and macro-structures and in-column orientation allows operation at comparably high linear velocities (~100 mm s-1) without excessive system backpressure. The ability to move fluid efficiently through the structure is complemented by the fact that the fiber physical structure is effectively nonporous with respect to the size of proteins, therefor there is no significant intrafiber diffusion. Ultimately, these factors combine for protein separations that are devoid of appreciable van Deemter C-term broadening.
Polypropylene C-CP fibers have been employed for fast protein separations in reversed phase (RP) mode. For hydrodynamic studies, important factors (injected mass and volume) affecting overload in terms of peak height, width and shape (asymmetry) were evaluated. For LC-MS application, a model mixture of ribonuclease A, cytochrome c, myoglobin and lysozyme were prepared in phosphate buffered saline (PBS) and urine matrices. The efficiency of the matrix removal was reflected in the near-identical qualitative and quantitative responses in both UV-Vis and MS detection. Later, a new, trilobal-shaped C-CP fiber was under developed to address the issue of poor A-term performance of the previous eight-channeled form. The trilobal geometry provides better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters.
Poly(ethylene terephythalate) (PET) C-CP fiber was used as the stationary phase for the separation of a synthetic protein mixture based on hydrophobic interaction chromatography (HIC). Optimum resolution and fast analysis times were achieved employing a steep gradient using higher linear velocities. The use of PET C-CP fibers in a HIC protocol was examined to isolate exosomes from a human plasma sample. Initial results demonstrated the ability to isolate exosomes with comparable yields and size distributions and on a much faster time scale when compared to traditional isolation methods, while also alleviating concomitant proteins and other impurities. And, 2D-HPLC method for characterizing mAb concentration and aggregation level, combining ProA purification, a novel injector-loop capture step (HIC processed by PET C-CP fiber), and aggregation determination by SEC is described. Advantages are seen in terms of limiting in-system mAb aggregation due to reduced low-pH solvent exposure, improved 2D chromatographic resolution, better monomer/aggregate ratio fidelity, and enhance quantitative figures of merit.
Recommended Citation
Wang, Lei, "Development of Capillary-Channeled Polymer (C-CP) Fiber as Stationary Phase for Biological Sample Application" (2020). All Dissertations. 2901.
https://open.clemson.edu/all_dissertations/2901