Scalable and intensified bioprocessing for virus-like particle manufacture

Liew, Mervyn Wing On (2012). Scalable and intensified bioprocessing for virus-like particle manufacture PhD Thesis, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.

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Author Liew, Mervyn Wing On
Thesis Title Scalable and intensified bioprocessing for virus-like particle manufacture
School, Centre or Institute Australian Institute for Bioengineering and Nanotechnology
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Anton Middelberg
Yap Pang Chuan
Total pages 128
Total colour pages 13
Total black and white pages 115
Language eng
Subjects 0904 Chemical Engineering
1003 Industrial Biotechnology
Formatted abstract
Process intensification through the engineering of highly selective and efficient bioprocesses is critical to drive the timely and economical delivery of biopharmaceutical products, such as virus-like particles (VLPs), to the market. VLPs are self-assembled from viral structural proteins, showing a high morphological resemblance to viruses but are non-infectious. VLPs have been used as efficacious vaccines against the native viruses from which they are derived, and increasingly as a display platform for foreign antigenic peptides. Utility of VLP vaccines to combat infectious disease at global scale is nevertheless hampered by high production costs inherent to inefficient conventional cell-based production systems. A superior manufacturing route based on efficient and rapid manufacture of VLPs using microbial in vitro VLP production, has been recently demonstrated. This approach involves expression of viral structural proteins using highly productive microbial hosts followed by cell-free assembly of VLPs under controlled environments. In this thesis, scalable and intensified upstream viral structural protein production and downstream VLP assembly and formulation bioprocesses were developed to enable this in vitro VLP manufacturing paradigm at industrial scale. This work demonstrates, for the first time, successful translation of the viral structural protein expression process from shake flasks to bioreactors, leading to a 15-fold improvement in volumetric yield. The expression of viral structural protein at gram-per-litre levels was achieved without compromise on product quality using a fed-batch high-cell-density cultivation process. The upstream yield improvements were paralled by the development of scalable and process-intensified downstream processes involving VLP assembly and formulation. An improved dilution VLP assembly method was designed, which reduced buffer consumption by 9-fold and increased product concentration by 5-fold. This dilution assembly method eliminated a process-inefficient concentration step, resulting in 22% absolute increase in VLP yield compared to conventional means. To reduce the number of downstream unit processes, intensification of the VLP assembly and formulation bioprocess was achieved through the use of a single diafiltration unit. The rate of assembly buffer exchange in a diafiltration process was found to significantly influence the yield and quality of assembled VLPs. By quantitatively investigating the distribution of VLP quartenary structures as a function of physicochemical condition change rate, a reactive VLP diafiltration process was engineered to produce high-quality VLPs. Taken together, the intensified bioprocesses developed in this thesis will have far-reaching benefits that enable low-cost, mass manufacture of VLP vaccines through the in vitro VLP manufacturing route.
Keyword Process intensification
Bioprocess design
Virus-like particles
Murine polyomavirus
Escherichia coli
Fed-batch cultivation
Downstream processing

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Created: Wed, 06 Feb 2013, 15:43:00 EST by Wing On Liew on behalf of Scholarly Communication and Digitisation Service