Date Degree Awarded

2012

Degree Type

Restricted to Claremont Colleges Dissertation

Degree Name

PHD in Applied Life Sciences

First Thesis/Dissertation Advisor

Matthew S. Croughan

Second Thesis/Dissertation Advisor

Tina Etcheverry

Third Thesis/Dissertation Advisor

Edward J. Perkins

Abstract

Current recombinant protein production systems require several months to develop. Existing systems fail to provide timely, flexible, and cost-effective therapies to protect against emergency mass-casualty infections or poisonings. As the identity of many new biological threat agents are unlikely to be known in advance, pre-emptive manufacturing and stockpiling of countermeasures cannot be performed. Preparedness for a biological catastrophe requires a radical solution to replace the current slow scale-up and manufacture of lifesaving medical countermeasures. Subunit vaccines and antibody fragments may be produced in bacteria, yeast, plant or insect cells. However, generation of full-length, human like glycosylated antibodies requires mammalian cell culture due to the cell’s ability to carry out complex assembly and processing. Although commercial cell culture methods for antibody production from stable gene expression have been substantially improved over the last 30 years, the time required to achieve full scale production for a new product is too long for a rapid, emergency response. An alternative method for rapid production of high quality antibody protein is transient gene expression.

Transient gene expression is an established, routine approach to small scale, researchgrade material of recombinant proteins. It is frequently used to generate gram quantities of material within weeks of lead target identification. In the past, transient gene expression has been considered for emergency production of large production of antibodies, but dismissed due to low titers, high DNA requirements, uncertain regulations, unavailable manufacturing capacity, and uncertain scale-up performance. If these barriers can be overcome in the next few years, emergency use of transient gene expression for production of life saving medical countermeasures would be a viable means to help protect our nation from biological attacks. The goal of this thesis is to investigate both the technical and operational feasibility of scaling up transient gene expression. In order to investigate the technical feasibility of such a method, a phenomenological understanding of transfection was developed for process characterization, process optimization and scale-up studies. Experiments in shaker flasks and lab-scale bioreactors interrogated a number of factors involved in the transfection process and identified an optimal design space for performing transfections (Chapters 2 and 3). Important factors that were identified include cell/DNA/PEI ratio, transfection incubation time, and agitation set points. Through this optimization process, the highest reported titer (>300 mg/L) in transient CHO production was achieved.

Experimental transfections also provided calibration metrics for phenomenological models of mass transfer in very large bioreactors. These models were used to investigate the potential of mass transfer limitations upon scale-up (Chapter 2). The results indicate that, with appropriate design of the agitation systems, including consideration of the impact of mass transfer of PEI/DNA complexes from the medium to the cells during the transfection stage, scale-up should be successful. In the final stage of experimentation, successful identification of scalable systems for aseptic liquid-cell separation eliminated other potential bottlenecks that may be encountered during scale-up (Chapter 3). A novel combination of existing technology generated simplified transfection protocols, which may be commercialized for alternative markets.

Operational feasibility was investigated through a survey of current manufacturing capacity for mammalian cell culture and their capabilities to provide meaningful emergency production of antibody countermeasures. Process simulation was conducted to analyze process flow, plant design, and cost for large scale production of both plasmid DNA (Chapter 5), a sub-component of transient gene expression, as well as antibody protein (Chapter 4). Simulations predicted that a 1,000-L fermentor would produce sufficient plasmid DNA at a cost of approximately $377/gram. This DNA could be utilized in a 200,000-L facility to produce between 32 and 1,274 kg of recombinant antibody. Experimentally validated transfection processes were then used to refine simulations. Subsequent simulation resulted in production of 197 kg within 3 months at a cost of 705 $/gram mAb. These simulations predict that largescale transient gene expression can provide sufficient lifesaving countermeasures if titer improvements are possible and can be successfully scaled to large bioreactors. This thesis demonstrates both the operational and technical feasibility of a successful large-scale transient gene expression platform for the production of full-length, human-like glycosylated antibodies as medical countermeasures for biological catastrophe. This approach is one critical component of our Nation’s arsenal of bio-defense capabilities.

Rights Information

© 2012 Christopher M. Warner

DOI

10.5642/kgitd/1

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