Temperature controlled, single column, continuous hydrophobic interaction chromatography of proteins

Brean, Alexander James ORCID: 0009-0007-2715-3233 (2025). Temperature controlled, single column, continuous hydrophobic interaction chromatography of proteins. University of Birmingham. Ph.D.

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Abstract

The increasing clinical relevance of mAbs has necessitated a substantial scale-up in biopharmaceutical production. To address this rising demand, numerous biopharmaceutical companies have invested in large-scale manufacturing facilities employing standardised platform processes (Shulka and Gottschalk, 2013; Shukla et al., 2017). Despite the operational advantages of continuous downstream processing methods, their adoption within the industry has been limited. The primary impediments include the excessive generation of equilibration, wash, elution, and regeneration buffers, contributing to inefficiency and waste. Additionally, the mechanical and software complexities of existing continuous chromatography systems render them costly and challenging to implement. Thus, there is a clear demand for innovative technologies that can facilitate continuous processing while offering sustainable advantages, such as reduced buffer and salt consumption.

This thesis seeks to advance a continuous chromatography solution, specifically through the development and application of the TCZR system. Unlike conventional continuous chromatography approaches that typically rely on multiple columns and intricate control mechanisms, the TCZR offers a streamlined alternative, employing a single-column system with temperature modulation. Prior applications of this technology have focused
on thermo-responsive cation exchange chromatography (Müller et al., 2013; Cao, 2015), and a thermally responsive protein A mutant (Ketterer et al., 2019). In this work, we extend the utility of the TCZR to hydrophobic interaction chromatography (TCZR-HIC), broadening its application in bioprocessing.

Chapter 2 documents the materials, methods, and instrumentation used in the below results chapters. As the TCZR, chromatography methods, and analysis are closely related between these chapters, these have been documented in a stand-alone chapter to avoid redundancy.

Chapter 3 presents a new application of the TCZR using HIC. In this work, TCZR-HIC is employed to purify bovine serum albumin isocraticly, meaning that the salt concentration remains constant during both binding and elution, which contributes to the process's sustainability. A local temperature decreases of ΔΦ = 30 °C was used to facilitate protein desorption, effectively decoupling the elution process from the mobile phase composition. This setup enables continuous TCZR-HIC with a single column, offering a more sustainable and simplified alternative to traditional multi-column systems.

Chapter 4 builds upon the principles established in Chapter 3, exploring the application of TCZR-HIC to a thermally stable and commercially significant mAb, Rituximab. The feasibility of utilizing TCZR-HIC in a bioprocessing context is evaluated with Rituximab as a model system. While the technique demonstrates potential, optimal results necessitate a greater temperature deference than the current TCZR instrumentation can
achieve.

Chapter 5 summarises the findings from the preceding studies and outlines possible directions for this research. Recommendations for enhancing the TCZR system are discussed, with a focus on overcoming the limitations identified in the current setup. To conclude, this thesis aims to elucidate the capabilities of TCZR-HIC through the purification of two model proteins, BSA and Rituximab. The research expands upon the foundational work by Müller and Franzreb, demonstrating continuous single-column purification and exploring the applicability of TCZR-HIC in an industrial bioprocessing context. The findings offer promising insights into the future of sustainable continuous chromatography technologies for biopharmaceutical production.

Type of Work:	Thesis (Doctorates > Ph.D.)
Award Type:	Doctorates > Ph.D.
Supervisor(s):	Supervisor(s) Email ORCID Thomas, Owen UNSPECIFIED orcid.org/0000-0001-8253-365X Overton, Tim UNSPECIFIED orcid.org/0000-0003-3050-2549 Franzreb, Matthias UNSPECIFIED orcid.org/0000-0003-3586-4215 Bracewell, Daniel UNSPECIFIED orcid.org/0000-0003-3866-3304
Licence:	All rights reserved
College/Faculty:	Colleges > College of Engineering & Physical Sciences
School or Department:	School of Chemical Engineering
Funders:	Engineering and Physical Sciences Research Council
Subjects:	Q Science > Q Science (General)
URI:	http://etheses.bham.ac.uk/id/eprint/16030

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