Date of Graduation

5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Chemical Engineering

Advisor/Mentor

Wickramasinghe, S. Ranil

Committee Member

Qian, Xianghong

Second Committee Member

Zydney, Andrew

Third Committee Member

Ford, David M.

Fourth Committee Member

Almodovar, Jorge

Keywords

Aggregation; Bioprocessing; Monoclonal antibody; Prefiltration; Process development; Virus filtration

Abstract

Virus filters are single-use devices that use a size-based separation process. In virus filters, contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filters are an essential component of the overall virus clearance strategy. Sections 1 and 2 of this dissertation provide an introduction and extensive review of monoclonal antibody (mAb) process development, where virus filtration is pivotal. In section 3, prefiltration studies were performed with an industrially relevant IgG1 type mAb using adsorptive and size-exclusion-based prefilters with different mechanisms of action. This mAb has an isoelectric point range of 7.1 to 8.0 and a molecular weight (MW) of 148 kDa. Decoupled prefiltration and virus filtration studies were conducted. We attempted to elute bound species from the membrane to identify them. Permeate fractions from the prefilters were introduced as feed fractions to a Planova BioEX (Asahi Kasei Medical, Tokyo, Japan) commercial virus filter for flux decay studies. Prefiltration and virus filtration studies were performed at different pH and ionic strength buffer conditions. By adjusting buffer conditions, and choosing prefilters with an appropriate mechanism of action, increased selectivity for foulant capture resulting in improved flux behavior during virus filtration could be achieved. Extensive characterization was also performed for the various filtration fractions to determine molecular species that increase fouling propensity in the virus filter and the efficacy of the different prefilters at removing these species. In section 4, prefiltration and flux decay studies on a Viresolve Pro (MilliporeSigma, Billerica, MA) as well as the Planova BioEX virus filter was performed with another industrially relevant mAb with an isoelectric point range of 5.95 - 6.55. The impact of excipients on mAb fouling behavior was determined. The impact of buffer pH was also evaluated with one pH condition below the isoelectric point (pI) of the mAb and another pH condition above the mAb pI. Decoupled prefiltration was performed to evaluate the impact of different types of prefilters on the filterability of this mAb. The pharmaceutical analysis system PA800 plus (SCIEX, Redwood City, CA) was also used to characterize the various mAb fractions from prefiltration and virus filtration. Dynamic light scattering (particle size analysis), size exclusion chromatography, SDS PAGE, capillary electrophoresis, and MALDI mass spectrometry were used for characterization. In section 5, a new technique of fractionating close molecular weight biomolecules was evaluated for virus clearance. The technique is known as internally staged ultrafiltration (ISUF), where layers of ultrafiltration membranes operate in stages to fractionate biomolecules based on differences in isoelectric points. The membranes of interest were the Pall Omega PES 300 kDa molecular weight cutoff (MWCO) flat sheet membrane, Pall Omega PES 100 kDa MWCO membrane, Millipore Ultracel 100kDa MWCO, and the Millipore Ultracel 30kDa MWCO. Virus clearance studies were performed using internally staged ultrafiltration membranes in skin and backing configurations. Section 6 is an overall conclusion for this work showing major findings and identifying areas for future study.

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