Date of Award

8-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemical and Biomolecular Engineering

Committee Chair/Advisor

Dr. Scott Husson

Committee Member

Dr. Mark Roberts

Committee Member

Dr. Marc R Birtwistle

Abstract

This thesis project describes the modification and evaluation of a new affinity membrane for rapid chromatographic purification of non-antibody proteins. The affinity membrane utilizes Im7/CL7 coupling technology developed by Dr. Vassylyev's lab at the University of Alabama Birmingham (UAB), licensed by TriAltus Bioscience. The behavior of the membrane was evaluated using purified CL7-tagged Cas9 as my model protein for static and dynamic binding capacity analysis.

Chapter one provides an overview on biopharmaceutical drug production process development. I discuss how protein drugs are produced, isolated, and purified from the cell supernatant after upstream phases are completed. Despite increasing demands for biologics, the isolation and purification of proteins from the cell supernatant, especially for non-antibody proteins where there is no universal affinity technology for purification, present major challenges for drug development and manufacturing. I highlight how new developments in downstream chromatography separation process, combined with multiple improvements in upstream manufacturing already in motion, will result in faster and higher volume production.

Chapter two describes the development of Im7 affinity membranes. I discuss the synthesis of the membranes using direct modification of a regenerated cellulose base membrane and the measurement of binding capacities for CL7 and Cas9 proteins. For synthesis, N, N’-disuccinimidyl carbonate was coupled to the membranes. Unreacted succinimide carbonyl groups were coupled to primary amines groups of diamino-dipropylamine spacers. The unreacted primary amine was reacted with iodoacetyl chloride. Im7 was covalently coupled to the membranes. Ligand incorporation was evaluated with UV and FT-IR spectroscopy analyses. The ligand density was measured to be 38 mg Im7/mL. The static binding capacities (SBC) were measured to be 15 mg CL7/mL and 30 mg CL7-Cas9 /mL.

Chapter three describes the development of Im7 affinity membrane using a polymer grafting approach and evaluation for the purification of Cas9. Surface-initiated atom transfer radical polymerization (ATRP) was used to graft 2-hydroxyethyl acrylate (HEA) from the membrane pores, growing polymer tentacles containing hydroxyl groups that were reacted with iodoacetyl chloride. Im7 was coupled covalently to the membranes through reaction with the iodoacetyl groups, as confirmed with spectroscopy analysis. The ligand density was measured to be 50 mg Im7/mL. For capturing analysis with purified proteins, the SBC were measured to be 20 mg CL7/mL and 50 mg CL7-Cas9 /mL. Dynamic binding capacities at 10% breakthrough (DBC) were measured to be 14.5 mg CL7/mL and 22 mg/mL CL7-Cas9.

Chapter four presents an overview of the upstream and downstream work done by Dr. Vassylyev's lab at UAB that includes cell culturing of the proteins and the development of the resin beads used for the purification of Cas9. The 4B resin ligand density was measured to be 35 mg/mL Im7. The SBC was measured to be 66.5 mg/mL CL7. The 6B resin ligand density was measured to be 18 mg/mL Im7. Its SBC was measured to be 38.8 mg/mL of CL7, and it had a DBC of ~8 mg/mL for purifying CL7-Cas9 from lysate.

Overall, Im7 affinity membranes produced by surface-initiated ATRP obtained better performance results than the membranes developed by the direct modification approach. Grafting HEA polymer chains from the membrane pores compensated for the smaller surface area than resin beads. It produced a higher Im7 ligand density compared to the TriAltus affinity resin beads and higher CL7-Cas9 capacity. Dynamic protein binding capacities remained constant after multiple runs of elution and regeneration. Therefore, the membranes can be reused for multiple runs without any production loss. Dynamic protein binding capacities for the membranes do not depend on flow rate and were higher compared to TriAltus resin beads.

Available for download on Thursday, August 31, 2023

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