Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

The biopharmaceutical market has seen tremendous growth over the past few decades as new recombinant proteins, monoclonal antibody-based therapeutics and gene therapies have been commercialized. After production in different mammalian, yeast and microbial expression hosts, each product must be concentrated and highly purified before clinical use. Protein A affinity methods provide a convenient and widely used platform for capturing and purifying monoclonal antibodies (mAbs), Fc-fusion proteins, antibody drug conjugates (ADCs) and bispecific antibodies (BsAbs). In this work, an attempt has been made to develop novel Protein A affinity ligands having higher binding capacity than the ligands on commercial resins. There is currently no similar platform technology for purifying increasingly important non-mAb protein therapeutics. Protein therapeutics such as single domain antibodies, single chain variable fragments, Fab fragments, interferons, epoetins, clotting factors, growth factors, insulin and insulin like analogues, enzymes etc. have traditionally been purified using multiple column steps based on ion exchange, hydrophobic interaction, mixed mode, and ceramic hydroxyapatite chromatography. These multicolumn approaches require significant optimization and often result in low product yields and recoveries. Thus, scalable and cost-effective alternatives to these currently used approaches are needed. Furthermore, these alternative methods should be convenient to use and allow for easy technology transfer between clinical drug discovery, process development and manufacturing. In this work, we propose the use of pH-sensitive self-removing affinity tags as a potential solution. Purification strategies based on self-removing and self-precipitating tags have been developed previously for laboratory scale protein purification. However, these methods utilize pH sensitive contiguous inteins which suffer from premature cleavage, resulting in significant product loss during p (open full item for complete abstract)

Committee: David Wood (Advisor); Eduardo Reategui (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Biochemistry; Chemical Engineering

Master of Science, The Ohio State University, 2021, Chemical Engineering

Bridging the gap between protein purification platforms at the laboratory scale and at the industrial scale remains a challenge, as there is no “one size fits all” technology that works for every protein. To date, only technology based on Protein A has managed to enter mainstream usage both in the lab and in industry, but it only works for monoclonal antibody and Fc fusions. Affinity chromatography can be used to purify most proteins, but a major downside of this is that they require the use of affinity tags – proteins purified with these tags must undergo further treatment to remove the tags, which is impractical at the industrial scale. Research into intein-based chromatography offers a possibility to get around this: engineered inteins can be used as a “self-cleaving” affinity tag that leaves a tagless target protein after elution. However, the rate at which inteins cleave themselves off a protein varies wildly depending on the target protein. This work aims to reduce that dependency by introducing a mutation known to reduce the intein's dependence on the +2 C-extein residue in the natural splicing version of the Npu intein into the engineered cleaving version to determine if it can also reduce its dependency on the +2 residue. The experiments performed in this work did not detect a significant difference in variance, however. In addition, it also appeared to slow down the overall cleavage kinetics, suggesting that the intein cleavage reaction is subject to different controls than the splicing reaction.

Committee: Andre Palmer (Committee Member); David Wood (Advisor) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2018, Chemical Engineering

Recombinant therapeutic proteins changed the world over 30 years ago when insulin, the first therapeutic protein, was approved. Since then, over 200 therapeutic proteins have been approved to treat a wide range of diseases from diabetes to immune disorders. Currently, there is no universal platform that can be used to purify any given target protein in a quick and inexpensive manner. The self-cleaving split intein tag technology remedies this issue by creating a universal platform that can purify any traceless, tagless target protein rapidly and economically. Chapter 2 discusses the combination of the split intein purification strategy with cell-free protein synthesis (CFPS) systems to reduce the time it takes to produce therapeutic proteins. With the cell-free systems, proteins can be produced in hours compared to days or even weeks. The combination of CFPS and the split intein tag technology has been utilized in the creation of a device to produce biologics on demand. The BioMOD device aims to produce a single-dose of any therapeutic protein within 24 hours, specifically with a military application in mind. Chapter 3 discusses the use of magnetic beads to mediate the split intein purification. Combining the split intein and magnetic beads creates a more efficient purification process that requires less buffer and set-up time. Four target proteins are used to demonstrate the applicability of the system. Chapter 4 discusses the regeneration of a commercially available resin that has been used to covalently immobilize the N fragment of the split intein using a thioester bond. Due to the commercially available resin having a high price point and the lengthy amount of time it takes to immobilize the N fragment, regeneration of the resin was necessary. A panel of buffers was screened to find the best regeneration buffer. Using the best buffer, a life cycle analysis was done using 20 regeneration cycles to show the resin could be regenerated multiple times. The develo (open full item for complete abstract)

Committee: David Wood (Advisor); Jeffrey Chalmers (Committee Member); Andre Palmer (Committee Member) Subjects: Chemical Engineering

Master of Science, The Ohio State University, 2016, Chemical Engineering

In biopharmaceutical industry, protein-based drugs have been rapidly growing over the past decade. Approximately 40%-90% of total cost of protein drug manufacturing is spent on downstream processing. Due to protein target complexity, multiple chromatography purification steps are required to obtain products with high purity. Dr. Wood's lab have been studying intein-mediated protein purification tag for years, one of the most significant discovery is the pH-sensitivity of intein cleaving reaction that result from zinc-binding motif. This work aims to investigate the mechanism of pH regulation on intein self-cleaving reaction and search for zinc-binding motif sequences with improved pH sensitivity.

Committee: David Wood (Advisor); Shang Tian Yang (Committee Member) Subjects: Chemical Engineering