Prof. Ken Marcus and collaborators at Clemson University are supported by the Chemical Measurement and Imaging Program (Division of Chemistry) and the Chemical and Biological Separations Program (Division of Chemical, Bioengineering, Environmental, and Transport Systems), with co-funding from the Experimental Program to Stimulate Competitive Research, to develop and characterize capillary-channeled polymer (C-CP) fibers as stationary phases for rapid processing of biomacromolecules. C-CP materials are amenable to a wide range of column formats (microbore to preparative) and offer advantages for macromolecular separations (relative to traditional porous-bead, membrane, and monolithic stationary phases), including low flow resistance, elimination of size-exclusion effects, much higher mass transfer rates, and high mobile phase velocities (not necessarily volume flow rates) and gradient rates without sacrificing chromatographic performance. These properties make C-CP fiber columns potentially useful as the second dimension for 2-D HPLC protein separations. Surface chemistries can be varied to affect separation while providing high levels of flexibility. Finally, the approach should prove relatively inexpensive for applications such as high speed and preparative separations (e.g., of proteins).
The studies involve a collaborative effort between research groups from the Department of Chemistry and the School of Materials Science and Engineering, both of which are affiliated with the Clemson University Center for Advanced Engineering of Fibers and Films (CAEFF). Successful completion of the work may lead to new commercial ventures in the textile and pharmaceutical industries in South Carolina and elsewhere. CAEFF programs expose graduate students to science and research philosophies across the science/engineering interface. The low cost and simplicity of constructing C-CP fiber columns will allow their use in newly designed undergraduate and graduate laboratory experiments illustrating the basic principles of protein chromatography.
Research efforts in this reporting period continued fundamental studies into the expanded use of capillary-channeled polymer (C-CP) fibers in analytical and preparative protein separations. In the first case, we look toward developing tools for downstream processing of fermentation media for the critical application of protein therapeutics. In the second,we seek to develop materials which permit rapid processing of complex samples with regards to proteomic applications. The first area of investigation involved the characterization of the C-CP materials for separations on the preparative scale. Specifically, two sets of studies were performed: 1) characterization of fiber porosity and its implications in affecting rapid separations and 2) determination of the protein loading capacity as a function of column packing density and sample throughput. The second area of investigation involved the development of surface modification strategies to affect highly selective protein separations. The first example involved the use of a simple immobilization strategy of the ligand molecule protein A, which has the capacity to selectively capture immunoglobulin G. This approach has promise toward reducing costs and processing times in the area of protein therapeutics. Secondly, a novel ligand family has been developed based on the chemisorption of head group-modified lipids as a means of creating robust and selective capture phases. This looks to be an incredibly versatile strategy that can be applied to all hydrophobic substrates. The final area of investigation deals with the use fo C-CP fibers to selectively extract proteins from complex media (e.g., urine and saliva) to alleviate sample matrix difficulties in subsequent mass spectrometric analysis. A micropipette tip format allows for rapid, straight processing, using a simple laboratory centrifuge to affect the seperations. Current emphasis is directed to the field of urinomics. Funding during this period supported 2-FTE graduate researchers, including two femaie graduate students. Those students generated data that resulted in one peer-reviewed journal publication, one currently in press, and two manuscripts which are currently under review. Finally, one of the supported graduate students, Ms. Abby Schadock-Hewitt recently completed 2013 NSF EAPSI Fellowship to study in the group of Prof. Conan Fee of the University of Canterbury, New Zealand. She began the 2-month program in June, but accepted a local fellowship to continue until the end of October. This collaboration will be an excellent resource for this group as Abby will come back with experience in use of preparative columns, while she will be teaching the Canterbury group her surface modification methods involving protein A and head group-modified PEG lipids.
