Processing of Virus-Like Particles

Daniel Lipin (2010). Processing of Virus-Like Particles PhD Thesis, School of Chemical Engineering, The University of Queensland.

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Author Daniel Lipin
Thesis Title Processing of Virus-Like Particles
School, Centre or Institute School of Chemical Engineering
Institution The University of Queensland
Publication date 2010-06
Thesis type PhD Thesis
Supervisor Anton PJ Middelberg
Linda HL Lua
Total pages 213
Total colour pages 19
Total black and white pages 194
Subjects 09 Engineering
Abstract/Summary A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.
Keyword virus-like particle
affinity chromatography
soluble aggregate
asymmetrical flow field-flow fractionation
transmission electron microscopy
electrospray differential mobility analysis
nuclear localisation
Additional Notes Colour pages: 4, 20, 25, 27, 33, 34, 44, 56, 84, 85, 86, 88, 89, 90, 168, 173, 175, 182, 187

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Created: Mon, 19 Jul 2010, 22:02:15 EST by Mr Daniel Lipin on behalf of Library - Information Access Service