This NSF award by the Chemical and Biological Separations program supports work by Professors Todd J. Menkhaus and Hao Fong at South Dakota School of Mines and Technology to fabricate, characterize, and evaluate an innovative adsorptive nanofelt made from electrospun cellulose and/or carbon nanofibers for the purification of large biomolecules such as proteins, viruses and DNA. Adsorptive membranes (also referred to here as adsorptive felts), have shown great promise for the downstream separation of biopharmaceutical products. Unfortunately, the binding capacity of large biomolecules on current commercial adsorptive felts is often low and can be limited by available surface area. In this project, innovative nanofelts made from cellulose and/or carbon nanofibers (with diameters in the range from 10 to 1000 nm) will be fabricated and evaluated for isolation of large biomolecules. These nanofiber nanofelts will provide a significantly elevated surface area to bed volume ratio (one to two orders of magnitude higher than conventional felts), which will offer much improved binding capacity, without compromising the desired hydrodynamic properties of high flow rates with low pressures. Nanofibers will be produced by the technique of electrospinning. Initially, preparation conditions of cellulose and carbon nanofibers will be defined, and the morphological and physical properties will be characterized. The nanofibers will then be functionalized with ionized chemical ligands, including state-of-the art three-dimensional grafting technologies, for adsorption of target molecules by ion exchange, and manufactured into nanofelts. Finally, static and dynamic adsorption capacity of a target molecule, system dispersion, and pressure profiles will be assessed for varying flow rates. Adsorption of both relatively large and small proteins, as pure solutions and as a mixture, will be evaluated to model the process and determine limiting factors of adsorption. This will provide insight into predicting optimum properties (i.e. pore size, felt thickness, and charge density) and operating conditions (i.e. flow rates) for the innovative nanofelt. From an applied perspective, results obtained in this project will provide improved technologies to reduce costs in the production of biopharmaceutical products, and will also be directly applicable to other separation fields such as environmental wastewater treatment. The multidisciplinary nature of the project will expose undergraduate and graduate students to cutting-edge technologies in both fabrications/evaluations and bioseparation applications of the electrospun adsorptive nanofelt, and prepare them for careers in the rapidly expanding nano and biotechnology industries. In addition, new problem-based learning modules in nano separation technologies will be incorporated into existing lectures/labs taught by the investigators (e.g., Design of Separation Processes, and Chemistry of Nanomaterials) to introduce important nano materials fabrication techniques and separations applications to a wide array of graduate and undergraduate students.
Production methods for both cellulose and carbon nanofibers have been greatly improved over currently reported protocols. Cellulose fibers with significantly smaller diameters and uniform morphology have been produced by careful control over properties of solutions and conditions of electrospinning. Additionally, the control over electrospinning, stabilization, and carbonization, and functionalization processes has allowed for the optimal production of carbon nanofibers with improved morphology and/or structure. The cellulose and carbon fibers have been further investigated with key discoveries found, such as: Cellulose nanofibers produced after functionalization by Atom Transfer Radical Polymerization (ATRP), provided over 15-times higher protein binding capacity than any other adsorption membrane that has been reported (commercial or in the scientific literature) for protein separations (including recent reports using ATRP with microfiber membranes). The elevated adsorption capacity does not detrimentally affect membrane permeance or adsorption kinetics. Thecarbon nanofibers proved to be very hydrophobic, even with effective surfacefunctionalization with anionic groups due to the high surface area. However,we were able to overcome the hydrophobicity challenge by introducing different additives to the adsorption solution, which then allowed for elevated bindingcapacities of protein onto the carbon nanofiber support with greatly reduced non-specific adsorption. We developed a novel mathematical model that is able to predict adsorption breakthrough from membrane adsorption devices. The new predictive model uses simple experimental data to account for adsorption capacity, adsorption kinetics, and the very important, but often neglected, hydrodynamic properties of the system. This led to the fundamental discovery that membrane adsorption systems for protein purification are universally limited by poor hydrodynamic flow distribution. New materials and devices are needed to account for this limitation. We developed a process and economic analysis model in (Bio) SuperPro Designer to evaluate the impact of replacing packed bed adsorption separations with membrane adsorption within a traditional monoclonal antibody process. We found that by implementing membrane adsorption in place of packed bed adsorption, the cost per unit mass of product could be reduced by 23% along with 50% reduction in total process time, and 40% less aqueous waste generation. Electrospun nanofiber mats/felts have been produced with metal chelating functionality on the fiber surfaces. These have been fully characterized to verify the complete incorporation of nickel or cobalt on the surface. Batch adsorption studies show that nanofiber-based immobilized metal affinity adsorption media is capable of binding two-times more protein, with equivalent yield and purity compared to commercial resin. In all cases cobalt was found to be more selective than nickel as an affinity ligand, but capacity was lower. During the duration of this NSF project, four graduate students, five undergraduate students, and one postdoctoral research scientist have been trained in the multi-disciplinary fields of biochemical separations and nanomaterials. One graduate student, who completed his research focusing on this NSF award, graduated in May, 2011, with a Master’s of Science in Chemical Engineering. His current employment is with Lonza Biotechnology in Maryland working in the downstream purification process development group. Two undergraduate students that has participated in the project from the summer of 2009 – summer of 2010 and the summer of 2011, respectively, graduated with BS degrees in Chemical Engineering and have initiated their graduate degree program in Chemical and Biological Engineering at the South Dakota School of Mines and Technology. Another undergraduate is continuing work on her dual degrees in Chemical Engineering and Chemistry, and chose to pursue an full time industrial position in the waste water treatment industry. The fourth undergraduate worked throughout the 2011/2012 academic year, along with two full time graduate students (one in materials science and one in chemical engineering) and the final undergraduate began working with the project in August, 2012. Furthermore, a postdoctoral research scientist, who has been partially working on this NSF project and has been an author/co-author of four peer-reviewed publications resulted from this project, will become a tenure-track assistant professor in the Joint School of Nanoscience and Nanoengineering at the North Carolina A&T State University and the University of North Carolina at Greensboro in January 2012. In addition to the contributions in human resource development directed toward research activities, during the duration of the project nearly 2500 K-12 students, including over 350 from Native American Indian Reservation schools, have been introduced to science/engineering fundamentals through hands-on interactive activities. In addition to the direct development of Human Resources, the project has resulted in the development of a new small business, Nanofiber Separations, LLC, which was founded in October, 2011 by Drs. Menkhaus and Fong. The company has been successful in receiving its first SBIR Phase I award and continues to seek opportunities to grow. Currently, the company employs five scientists/engineers, all of whom have advanced degrees in science or engineering.
