Date of Award

8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Chemical Engineering

Advisor

Dr. Scott M. Husson

Committee Member

Dr. Charles Gooding

Committee Member

Dr. Douglas E. Hirt

Committee Member

Dr. Igor Luzinov

Abstract

This Dissertation centers on the surface-modification of macroporous membranes to make them selective adsorbers for different proteins, and the analysis of the performance of these membranes relative to existing technology. Traditional chromatographic separations for the isolation and purification of proteins implement a column packed with resin beads or gel media that contain specific binding ligands on their exposed surface area. The productivity of this process is balanced by the effective use of the binding sites within the column and the speed at which the separation can take place, in addition to the need to maintain sufficient protein purity and bioactivity. Because of the nature of the densely packed columns and, in the case of resin columns, the limited access to the binding sites internally located within the resin, the operating speed of this separation process may be constrained by mass-transfer and pressure limitations. Other constraints include the time-intensive measures taken to properly pack the columns, and the challenges associated with scaling chromatography columns to industrial-sized processes. Because of the excellent selectivity, chromatography processes are the workhorse for biopharmaceuticals drugs, and other plant- and animal- based protein products. Thus, there are many markets that could benefit by improvements to this technology. My strategy focused on modifying porous membranes with surface-initiated atom transfer radical polymerization (ATRP) to grow polymer chains containing functional groups that target three different protein-ligand interactions for three different types of chromatography: cation-exchange, carbohydrate affinity, and Arginine-specific affinity chromatography. Although each of these types of separation has different challenges and different possibilities for impact among their unique applications, they all have the common need for a stationary phase platform with the potential for fast separations and specific interactions. The common approach used in these studies, which is using membrane technology for chromatographic applications and using ATRP as a surface modification technique, will be introduced and supported by a brief review in Chapter 1. The specific approaches to address the unique challenges and motivations of each study system are given in the introduction sections of the respective Dissertation chapters. Chapter 2 describes my work to develop cation-exchange membranes. I discuss the polymer growth kinetics and characterization of the membrane surface. I also present an analysis of productivity, which measures the mass of protein that can bind to the stationary phase per volume of stationary phase adsorbing material per time. Surprisingly and despite its importance, this performance measure was not described in previous literature. Because of the significantly shorter residence time necessary for binding to occur, the productivity of these cation-exchange membrane adsorbers (300 mg/mL/min) is nearly two orders of magnitude higher than the productivity of a commercial resin product (4 mg/mL/min). My work studying membrane adsorbers for affinity separations was built on the productivity potential of this approach, as articulated in the conclusion of Chapter 2. Chapter 3 focuses on the chemical formulation work to incorporate glycoligands into the backbone of polymer tentacles grown from the surface of the same membrane stationary phase. Emphasis is given to characterizing and testing the working formulation for ligand incorporation, and details about how I arrived at this formulation are given in Appendix B. The plant protein, or lectin, Concanavalin A (conA) was used as the target protein. The carbohydrate affinity membrane adsorbers were found to have a static binding capacity for con A (6.0 mg/mL) that is nearly the same as the typical dextran-based separation media used in practice. Binding under dynamic conditions was tested using flow rates of 0.1−1.0 mL/min. No bound lectin was observed for the higher flow rate. The first Damkohler number was used to assess whether adsorption kinetics or mass transport contributed the limitation to conA binding. Analyses indicate that this system is not limited by the accessibility of the binding sites, but by the inherently low rate of adsorption of conA onto the glycopolymer. The research described in Chapter 4 focuses on reaction chemistry experiments to incorporate a phosphonate-based polymer in the membrane platform to develop a new class of affinity adsorbers that function based on their affinity for Arginine (Arg) amino acid residues. The hypothesis was that benzyl phosphonate-containing functional polymers would form strong complexes with Arg-rich proteins as a result of multivalent binding. Introducing a new class of affinity membranes for purification of Arg-rich and Arg-tagged proteins may have an impact similar to the introduction of immobilized metal ion affinity chromatography (IMAC), which would be a significant achievement. Using Arg-tags would overcome some of the associated drawbacks of using metal ions in IMAC. Additionally, some cell penetrating peptides are said to be Arg-rich, and this would be a convenient feature to exploit for their isolation and purification. Lysozyme was used as a model Arg-rich protein. The affinity membranes show a static binding capacity of 3 mg/mL. This Dissertation is a demonstration of the potential and challenges associated with development of membrane materials for cation-exchange chromatography for protein capture, affinity purification of conA and other lectins, and affinity separations targeting Arg-rich regions of proteins and peptides.

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