Date of Graduation

12-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Xianghong Qian

Committee Member

Ranil Wickramasinghe

Second Committee Member

Kyle Quinn

Third Committee Member

Morten Jensen

Keywords

Biopharmaceutical, Bioseparation, Downstream Process, Membrane, Virus Filtration

Abstract

Virus filtration is an integral part of the downstream purification of mammalian cell culture-derived biotherapeutics to assure the viral safety of the products. Virus filtration membranes remove viruses based on a size-exclusion mechanism. Commercial parvovirus filers possess unique membrane structure and are designed to remove smaller non-enveloped parvoviruses with size 18-26 nm. However, some filters face issues, such as pre-mature fouling, the decline of filtrate flux, and reduction in virus retention. This doctoral dissertation focused on identifying the factors that influence the filtrate flux and the virus retention capability of commercial virus filters. The effects of solution pH and ionic strength, buffer type, protein molecules, and filter properties on virus filters' performance are investigated.

In Chapter 2, virus filtration was performed for an Fc-fusion protein using three commercial virus filters in solutions with varying buffer type and salt concentrations. A combined pore blockage and cake filtration model successfully described the three filters' fouling behavior in all the solution conditions. The model indicates that the blockage of membrane pores by larger particles (e.g., virus and protein aggregates) occurs in the initial stage of the filtration, followed by a cake filtration where particles accumulate on the membrane surface. Both the pore blockage rate and the rate of increase of protein layer resistance over blocked pores are affected by the solution conditions and membrane properties.

In Chapter 3, an Food and Drug Administration (FDA) recommended model virus, minute virus of mice (MVM) was spiked into the solution of three different protein molecules under various solution conditions, then filtered with three commercial virus filters. The log reduction values (LRV) were determined to systematically evaluate the effects of solution pH, salt concentration, buffer species, protein molecules' properties, cumulative viral loads, and the process interruption on the virus retention capability of the virus filters. The results showed that some virus filter is more prone to virus breakthrough than the others, both during the filtration and after a process interruption. In some cases, a higher flux is associated with a more significant LRV decline.

The diffusion interaction parameter, ??, is an indicator of colloidal and thermal stability of proteins in solution and a negative ?? represents the presence of attractive intermolecular forces. The ?? of a monoclonal antibody was determined in various solution conditions and was found to be affected by the pH, ionic strength, and the buffer species. Besides, a more negative ?? value is associated with a lower average relative flux of a commercial virus filter. These results indicate that ?? can be potentially used as an indicator of virus filter performance. The comparison between a two-layer membrane and a single-layer module of the same membrane was made to investigate the cause for their difference in virus retention. Fluorescent labeling of the virus and the protein was done; confocal imaging of the membranes after filtration was done as well to visualize the location of the captured virus and protein within the membrane.

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