Date of Award

2020

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Chemical Engineering (PhD)

Administrative Home Department

Department of Chemical Engineering

Advisor 1

Caryn Heldt

Advisor 2

Julia King

Committee Member 1

Tony Rogers

Committee Member 2

Warren Perger

Abstract

Viruses are responsible for many human diseases that lead to suffering and death. Acquired immunodeficiency syndrome (AIDS), influenza, and coronavirus disease 2019 (COVID-19) have claimed the lives of millions of people. Two methods have been created to reduce the suffering caused by these diseases. The first is the manufacturing of vaccines that prevent disease and the second is the development of virus detection tests that lead to treatment. The work summarized in this dissertation discusses the development of a continuous virus purification process using an aqueous two-phase system for use in vaccine manufacturing. Additionally, two novel virus detection methods were developed using graphene biosensors and a gold nanoparticle aggregation assay.

This dissertation contains results obtained from a novel continuous viral particle purification technique. The biomanufacturing industry would greatly benefit by switching from traditional batch processes to continuous processing. The reduction in costs and increase in productivity of continuous unit operations are driving the desire for continuous biomanufacturing processes. Switching to continuous processing would eliminate the current downstream processing challenges of viral-based biotherapeutics including limited yield and throughput. The method developed here was a chromatography-free separation technique aimed to increase throughput, purity, and yield of viral particles. The technique utilized an aqueous two-phase system (ATPS) to partition viral particles to a different phase than contaminant proteins. The ATPS consisted of poly(ethylene glycol) (PEG) and sodium citrate to separate viral particles from contaminant proteins from cell culture media. The ATPS was scaled-up from 1 g batch systems and run continuously for porcine parvovirus (PPV) and human immunodeficiency virus-like particles (HIV VLPs). Results from the purification of both viruses showed >70% recovery with a majority of the contaminating proteins and DNA being removed. This system has the potential to be further scaled-up for full-scale continuous biomanufacturing of vaccines.

This dissertation also contains results from the development of two viral detection methods. The first detection method used a graphene ink-based paper biosensor to electrochemically detect the presence of biomolecules. These graphene biosensors were created with the goal of being lab-on-a-chip devices for rapid detection in low-resource areas without the need of expensive laboratory equipment. The biosensors initially showed promise for portable, inexpensive, and rapid detection of proteins via electrical resistance changes at the sensor surface, but ultimately could not be applied to virus particles. The second detection test utilized osmolyte-induced gold nanoparticle (AuNP)/virus complex aggregation to detect whole viral particles. The AuNP aggregation assay was previously developed to detect two model viruses, PPV and bovine viral diarrhea virus (BVDV), using one size of AuNPs. The AuNP assay was capable of nanomolar level detection of both viruses. Optimization of this previously established virus detection method was performed by utilizing various sizes of AuNPs to lower the limit of detection (LOD). Results from using other sizes of AuNPs showed no improvement to the LOD.

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