Date of Graduation
Master of Science in Cell & Molecular Biology (MS)
Second Committee Member
Robert Beitle, Jr.
biotherapeutics, monoclonal antibodies (mAbs), prefiltration, machine learning
A virus filtration step is integral to the manufacturing of biopharmaceuticals to ensure viral safety. A virus filter is a single-use device that uses a size-based separation process and has a unique structure with minimal defects, so that contaminating virus particles cannot pass through the membrane pores, while therapeutic molecules can. The development of novel antibodies (Abs), including significant increases in product titers, is frequently challenged by virus filter fouling, making a better understanding of the underlying mechanisms essential. This thesis focused investigating of the effect of prefilter types, buffer type and salt content on virus filtration performance. The impact of the protein-protein interactions and their aggregates on the filtrate flux during virus filtration of highly concentrated mAb solutions are also investigated. In Chapter 2, prefiltration and flux decay studies were performed with a different concentration of polyclonal human immunoglobulin G (IgG) using two commercially Prefilters (Viresolve® pre-filter and Planova 75N) and virus filters (Viresolve® Pro and Planova BioEx). The fouling behaviors of the virus filters were strongly affected by the concentration of the feed solution and protein oligomers. The prefilters did not provide much benefit in improving the filtrate flux at high concentrations. Filtration using Planova BioEX in two runs without prefiltration shows less fouling and significant flux improvement in the second run and the application of post-filtration buffer flush with different conditions results in higher flux recovery in the second run than the first run. Size exclusion chromatography (SEC) analysis for IgG solution in different buffer conditions revealed that the aggregate concentration increases slightly as the IgG concentration increases. Results using dynamic light scattering (DLS) in the same buffer conditions shows that the mean hydrodynamic diameter increases as the increase of protein concentration and the protein tends to have attractive interactions in both buffer conditions. This IgG has a high molecular weight (250 kDa) and it is moderately hydrophobic. In Chapter 3, virus filtration was performed with an industrially monoclonal antibody (mAb C) with different concentrations up to 50 g/L in different buffer conditions using Viresolve Pro virus filter and adsorptive prefilters. This mAb was subjected to decoupled prefiltration to evaluate how different types of prefilters affected its filterability. Filter fouling is found to be strongly affected by the product concentration and the presence of aggregates. An analysis of size exclusion chromatography (SEC) showed that the presence of large amount of high molecular weight species considered as irreversible aggregates correlates with irreversible fouling that caused reduced mAb throughput and filter fouling. Results using dynamic light scattering (DLS) in different buffer conditions shows that the mean hydrodynamic diameter increases as the increase of protein concentration and the protein tend to have a stronger tendency to aggregate in tris buffer compared to sodium phosphate buffer. The pharmaceutical analysis system PA800 plus was also used to characterize the various mAb fractions from prefiltration and virus filtration. In Chapter 4, an overall conclusion for this work showing major findings and identifying areas for future study.
Saab, I. A. (2023). Investigation the Plugging Behavior of Virus filters. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5059