EFFECT OF ELECTROSTATIC INTERACTIONS DURING PROTEIN ULTRAFILTRATION: EFFECTS OF LIGAND CHEMISTRY AND PROTEIN SURFACE CHARGE DISTRIBUTION
Open Access
- Author:
- Mardoukhi Rohani, Mahsa
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 03, 2011
- Committee Members:
- Professor Andrew L Zydney, Dissertation Advisor/Co-Advisor
Andrew Zydney, Committee Chair/Co-Chair
Ali Borhan, Committee Member
Themis Matsoukas, Committee Member
Arnold Anthony Fontaine, Committee Member - Keywords:
- Ultrafiltration
Electrostatic Interactions
Membrane Filtration
Protein - Abstract:
- The production of high value recombinant proteins requires robust, cost-effective, and high-resolution purification methods that can provide high yield and purification. Although ultrafiltration (UF) was originally viewed as a purely size-based separation process, it is now well established that the rate of protein transmission is strongly affected by electrostatic interactions. The overall objective of this thesis was to develop a more fundamental understanding of the role of electrostatic interactions in determining the transport and separation characteristics of a variety of electrically charged ultrafiltration membranes produced using a range of negative, positive, and zwitterionic ligands. A series of novel positively-charged ultrafiltration membranes were generated by covalent attachment of ligands having similar size but containing different numbers of primary, secondary, and quaternary amines. Protein transmission through these membranes was well correlated with the apparent zeta potential of the membrane, irrespective of the detailed ligand structure. The most strongly charged membrane provided more than 40-fold selectivity for the separation of cytochrome c from a neutral dextran with similar hydrodynamic radius. Experimental data were obtained for transmission of neutral, basic, and acid proteins using a series of zwitterionic ultrafiltration membranes generated by covalent attachment of small zwitterionic ligands to a base cellulose membrane. The sieving coefficients well correlated with the product of the surface charge densities of the protein and membrane, consistent with a partitioning model accounting for electrostatic effects. The apparent zeta potential of these zwitterionic membranes could be described using the pKa values of an analog of the zwitterionic ligand accounting for the conversion of the primary amine to a secondary amine through the chemical linkage. The zwitterionic membranes showed minimal fouling even under conditions where the protein and membrane had opposite charge, making them attractive in bioprocessing applications. The effects of protein surface charge distribution were studied using model proteins with similar size but different amino acid composition and surface charge distribution. The results demonstrated that the protein sieving coefficient is determined almost entirely by the net surface charge density of the protein, irrespective of the detailed distribution of charge groups over the protein surface. This behavior is very different than that seen in ion-exchange chromatography where binding is determined by the presence of localized charge patches. The separation characteristics of the charge-modified membranes were also studied using protein charge ladders, a set of covalently modified derivatives of a single protein having different net charge. The selectivity increased with increasing membrane zeta potential, with a sharp decline in the sieving coefficient when the variant charge exceeded a critical value. These results have implications in the use of charge-modified membranes for the separation of protein variants in large-scale downstream processing.