Author ORCID Identifier:
https://orcid.org/0009-0008-9999-7117
Date of Graduation
8-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Engineering (PhD)
Degree Level
Graduate
Department
Biomedical Engineering
Advisor/Mentor
Qian, Xianghong
Committee Member
Daniel Strauss
Keywords
Biotechnology; Confocal Microscopy; Ion Exchange Chromatography; Monoclonal Antibody; Virus Clearance; Virus Filtration
Abstract
Ensuring viral safety is critical in the production of biotherapeutics derived from mammalian cell cultures. Virus filtration (VF) is a critical step in downstream bioprocessing, designed to remove potential viral contaminants while maintaining product yield and quality. However, virus retention efficiency can be influenced by process conditions, protein interactions, and membrane fouling mechanisms. This dissertation explores the mechanisms of virus retention in commercial virus filters and examines the effects of process pauses and protein fouling. In addition to virus filtration, this dissertation also evaluates Anion exchange Chromatography (AEX), which evaluates alternative AEX, such as AEX membrane and multimodal AEX membrane. The study used confocal laser scanning microscopy to visualize virus retention, providing direct insights into the retention behaviors of Minute Virus of Mice (MVM) in various feed conditions. Findings from this research offer better mechanistic understanding into virus filtration and alternative virus clearance methods, contributing to enhanced process robustness in biopharmaceutical manufacturing. In Chapter 2, virus filtration was conducted to investigate the impact of process pauses and different protein solutions on virus retention behavior within Planova™ series virus filters (20N, S20N, and BioEX) under constant flux (75 LMH) with high throughput. Confocal microscopy was used to image the fluorescently labeled MVM and ImageJ was used to plot the retention profiles. Each membrane with distinct membrane structures shows different virus retention profiles. The results demonstrate that process pauses effects promote deeper viral migration within membranes due to Brownian motion by diffusion. BSA-induced fouling significantly shifts and broadens retention profiles, indicating severe fouling effects and pore blockage. Conversely, mAb feeds, with minimal aggregates, result in minor retention shifts, primarily via adsorption-related pore narrowing. Building upon the findings presented in Chapter 2, the next Chapter 3 systematically explores the impact of flow dynamics (constant flux vs. constant pressure) on virus retention across three virus filters. Filtration experiments were performed under constant pressure at 0.1 MPa and constant flux conditions identical to those described previously. Confocal microscopy and retention profiles revealed that virus retention inside the membrane was significantly influenced by convective and diffusive transport mechanisms, which can be quantified by Peclet number. Under constant pressure, membrane fouling, particularly in the BSA feeds, worsens the localized virus retention through mechanisms of pore blockage and diffusion. This research provides mechanistic understanding into the impacts of membrane structure, filtration dynamics, protein feeds with different aggregates level and process pause on the virus retention behaviors. In addition to virus filtration, alternative virus clearance strategies, AEX, were investigated in this dissertation. Membrane chromatography provides notable advantages over traditional packed-bed chromatography, including higher achievable flow rates and reduced mass transfer limitations. Chapter 4 compares the MVM clearance performance of membrane adsorbers and conventional resin-based AEX columns in different protein feed solutions. AEX membrane exhibited better virus removal efficiency across varying flow conditions and maintained robust clearance even with feeds containing high impurity levels, demonstrating its promising potential as a viable alternative to traditional chromatography. Furthermore, recognizing the limitation posed by high salt concentrations on traditional AEX chromatography, chapter 5 evaluates multimodal membrane chromatography for virus removal during monoclonal antibody purification. The results highlight that multimodal membranes significantly enhance virus retention compared to conventional AEX membranes, thereby providing an effective, robust, and scalable approach for virus clearance.
Citation
Xu, W. (2025). Mechanistic Understanding of Virus Retention during Virus Filtration for the Downstream Purification of Therapeutic Proteins. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5843
