Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemical and Biomolecular Engineering

Committee Chair/Advisor

Dr. Scott M. Husson

Committee Member

Dr. Marc R. Birtwistle

Committee Member

Dr. R. Kenneth Marcus

Committee Member

Dr. Sarah Harcum


This dissertation explores development and characterization of membrane chromatography tools for downstream purification of therapeutic biomolecules. Convective technologies, particularly membrane chromatography, are emerging in the field of bioseparations as an alternative to resin chromatography due to their improved throughput capability. On the forefront of emerging membrane technologies are multimodal and Protein A membrane adsorbers. The overarching theme of this dissertation research was to investigate advances in these membrane chromatography tools. The primary objective was to develop and characterize novel Protein A membrane adsorbers. A secondary objective was to characterize newly commercialized Protein A and multimodal anion exchange membrane adsorbers.

Two chapters address new ways to synthesize Protein A membranes utilizing alkyne azide click chemistry. In one study, Protein A membranes were synthesized utilizing a combination of copper (I) alkyne azide click chemistry and Atom Transfer Radical Polymerization (ATRP). Regenerated cellulose membranes, functionalized using a copolymer containing propargyl methacrylate and polyethylene glycol methacrylate, were reacted with azide-conjugated Protein A. The results show that the performance of these novel Protein A membrane adsorbers depends on copolymer composition and initiator concentration. Like other adsorptive membranes, the Protein A membranes synthesized by this click chemistry approach maintained dynamic binding capacities for human immunoglobulin G (hIgG) protein that are independent of flow rate.

In a second study, Protein A membrane adsorbers were synthesized using copper-free dibenzocylcoctyne (DBCO) click chemistry. The synthetic approach included incorporation of DBCO groups into Protein A via N-hydroxysuccinimide ester bioconjugation; preparation of azide functionalized membranes using disuccinimidyl carbonate functionalization of hydroxyl groups on the membrane supports, followed by reaction with an azido-PEG3-amine molecule; and finally a click reaction between DBCO-conjugated Protein A and the azide-functionalized membrane surface. Conjugation of Protein A ligand was performed as a function of DBCO-PEG5-linker. Synthesized Protein A membranes, characterized using static and dynamic binding capacity of hIgG, showed that the molar ratio of hIgG to immobilized Protein A can be affected by covalent conjugation of Protein A. Again, dynamic binding capacities were independent of flow rate.

In a third study, an in-depth characterization of commercial Protein A membrane adsorbers was performed. The new Protein A membrane adsorbers included Purilogics Purexa™-PrA, Cytiva HiTrap Fibro™ PrismA, Gore® Protein Capture Device, and Sartorius Sartobind® Protein A. Several process relevant metrics were evaluated, including: dynamic binding capacity, equilibrium binding capacity, elution volume, permeability, yield, and impurity clearance. Results showed notable advances in binding capacity values, with Purilogics Purexa™-Pr A and Cytiva HiTrap Fibro™ PrismA being able to reach ~70 mg/mL membrane at processing speeds as fast as 5 s residence time. On the other hand, elution volumes for these Protein A membranes showed flow-dependent tailing at short residence times. The Gore® Protein Device showed the highest dependence of binding capacity on flow rate, however, the elution volume was the smallest of all devices tested and was independent of flow rate. All membranes showed different impurity clearance profiles and yield values; but, generally, all were able to reach at least 1.5 to 2 log reduction values of host cell proteins and double stranded DNA with at least 80% yield. This study showed that state-of-the-art Protein A membrane adsorbers have much improved binding capacities. The next frontier of development is in minimizing the flow distribution to improve elution volumes.

In the last study, a novel multimodal anion exchange membrane, Purilogics Purexa™-MQ, was characterized. Results show that incorporation of anion exchange and hydrophobic ligands improved salt tolerant binding capacity over other commercial resins and membranes. Additionally, there were differences in product-related impurity clearance for this product compared to other products. Under the conditions tested, Purexa™-MQ was capable of clearing dimers and high molecular weight species where conventional anion exchangers do not. This is an important advance for using conductivity as a variable to tune separation selectivity with membrane adsorbers, without having to sacrifice binding capacity.

Author ORCID Identifier




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