Date of Award
Doctor of Philosophy (PhD)
Husson, Scott M
Christensen , Kenneth A
Gooding , Charles H
Hirt , Douglas E
Wickramasinghe , Ranil
This doctoral research focuses on the design, development and characterization of advanced ion-exchange membranes and their performance evaluation as process chromatography media for downstream bioseparations. Chromatography is a widely used unit operation in the biopharmaceutical industry for the downstream purification of protein therapeutics. The rapid developments in biotechnology and the pharmaceutical potential of biomolecules have increased the worldwide demand for protein therapeutics dramatically. Considering that 50−90% of the total cost of bioprocesses is due to the downstream recovery and purification, high-productivity and high-resolution separation techniques that will enable cost-effective production are essential to the biopharmaceutical industry. In recent years, membrane chromatography has been promoted as a promising alternative to more conventional packed-bed resin chromatography. Although the potential for membrane chromatography is great, the historically lower binding capacity of membranes compared to resin media has limited its broad implementation. Therefore, primary objectives of this dissertation were to prepare advanced weak and strong anion-exchange membranes with ultrahigh and completely reversible protein binding capacities and to demonstrate the high-throughput and high resolution that these membranes enable in the separation of a target protein from a complex media (cell lysate).
The research presented here pertains to the use of atom transfer radical polymerization (ATRP) to prepare surface-modified weak and strong anion-exchange membranes for chromatographic bioseparations. Surface-initiated atom transfer radical polymerization (ATRP) was used to graft poly(2-dimethylaminoethyl methacrylate), (poly(DMAEMA)), and poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), (poly(MAETMAC)), nanolayers from the internal pore surfaces of commercial regenerated microporous membranes. Characterization of physicochemical and performance properties of newly designed, surface-modified membranes was performed using various analytical techniques.
The central theme of my research was to investigate how polymer architecture influences the separation performance properties of surface-modified ion-exchange membranes. In one study, the grafting density and average molecular weight of polymer chains grown from the membrane pore surfaces were varied independently and optimized to prepare weak anion-exchange membranes with ultra-high and completely reversible dynamic binding capacity. The effects of polymer grafting density, average molar mass of polymer and linear flow velocity on the dynamic binding capacity were studied. This study yielded weak anion-exchange membranes with very high volumetric protein binding capacities (static binding capacity∼140 mg BSA/mL and dynamic capacity ∼130 mg/mL) at high linear flow velocities (>350 cm/h) and relatively low transmembrane pressure drop (<3 bar). In a second study, a systematic evaluation was performed on the role of polymer molecular architecture on the separation performance of strong anion-exchange membranes. Anion-exchange membranes with different polymer chain densities were prepared using surface-initiated ATRP. The effect of polymer chain density, and, thus the, degree of polymer grafting, on the mass transfer resistances and accessibility of large biopolymers (IgG and DNA) was studied. From this detailed study, I have prepared a unique protocol to design strong Q-type anion-exchange membranes with unusually high volumetric protein binding capacities (dynamic binding capacity ∼140 mg IgG/mL and ∼27 mg DNA/mL) at high linear flow velocities (>190 cm/h) and relatively low transmembrane pressure drop (<3.5 bar). Overall, findings from my Doctor of Philosophy (PhD) studies strengthen the argument that membrane chromatography can be a higher capacity and higher throughput process than resin chromatography.
Finally, I evaluated the protein separation performance of my newly designed anion-exchange membrane adsorber and compared it to a commercial membrane adsorber and resin column. One aspect of this study was to compare the protein separation performance of membrane chromatography with resin column chromatography. Anion-exchange chromatography was used under salt-gradient and pH-gradient elution to separate anthrax protective antigen protein from periplasmic Escherichia coli lysate. Overall, this part of the work demonstrates that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher (15 times higher than widely used resin column) volumetric throughput.
Bhut, Bharatkumar, "DESIGN OF ADVANCED ION-EXCHANGE MEMBRANES AND THEIR PERFORMANCE ASSESSMENT FOR DOWNSTREAM CHROMATOGRAPHIC BIOSEPARATIONS" (2010). All Dissertations. 594.