(323d) Towards the Large-Scale Chemical Self-Assembly Processing of Virus-like Particles

A virus-like particle (VLP) is an assembly of viral structural proteins into a protein coat which does not contain nucleic acid required for the reproduction of a virus, and therefore is non-infectious. There has been substantial research into developing VLP technology as a novel platform for gene therapy [1] and drug delivery[2]. However, the current market demand for VLPs is for the manufacture of vaccination products. Several types of VLPs have been shown to be very immunogenic [3] and are now being developed for vaccines in humans or animals. For example, a prophylactic VLP vaccine based on the structure of human papillomavirus (HPV) is now being commercialized by Merck and Company (NJ, USA). This HPV vaccine has proven to give 100% immuno-protection against cervical cancer, precancerous lesions and genital warts in young women[4]. Such a high level of protection for a broad-coverage vaccine is rare, highlighting the potential health benefits achievable through VLP technology. Furthermore, vaccines for emerging diseases such as pandemic influenza [5] and gastroenteritis [6] may also be developed to meet urgent needs of the society by taking advantage of the versatility of this platform technology. However, this breakthrough technology also poses new challenges in biomolecular engineering which necessitate detailed engineering studies into the scale-up of VLP manufacture[7].

The aforementioned HPV vaccine was manufactured through the in vivo self assembly of VLPs in Saccharomyces cerevisiae [8]. Other eukaryotic expression systems such as mammalian [9-11] and insect [12, 13] cells have also been adopted for the in vivo production of VLPs at laboratory scale. After expression of VLPs in vivo, additional in vitro disassembly/reassembly steps are often required to remove contaminants encapsulated within the VLPs and to improve the homogeneity and stability of VLPs[14, 15]. However, there was evidence of product heterogeneity even after such additional purification steps, and product loss associated with these additional purification steps can be as high as 20%[7]. An alternate route of manufacturing VLPs through in vitro chemical self-assembling processing has been presented [16-18]. For this processing route, the precursors for self-assembly of VLPs can be manufactured with high precision and purified to near homogeneity, and subsequent self-assembly reactions can be directed and controlled to give consistent products.

In this study, the major structural protein, VP1, of murine polyomavirus was expressed in Escherichia coli as a GST fusion protein. The recombinant protein was purified with affinity chromatography and cleaved with thrombin for the removal of the GST fusion tag. After subsequent purification, VP1 protein with 90% homogeneity was obtained and assembled into polyomavirus-like particles with product yields up to 90%. We have optimized the expression and cleavage conditions for the recombinant VP1 protein, and modeled affinity chromatography to optimize the purification of recombinant fusion proteins. Furthermore, by coupling asymmetrical flow field-flow fractionation (AFFF) with multiangle light-scattering (MALS) technique, we have developed an efficient and systematic approach to optimize and validate the processing steps for the manufacture of VLPs through self –assembly reactions.

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