(70c) Simple and Efficient Downstream Bioprocessing of Virus-Like Particles Using Cell Free Reactors

Virus-like particles (VLPs) are now being developed as a new class of vaccines that provides unprecedented safety and extraordinary immunoprotection compared to traditional vaccines such as live attenuated viruses. This was demonstrated in a recent clinical trial for Gardasil, a VLP based vaccine commercialized by Merck (West Point, PA, USA), which showed that that the vaccine is 100% effective in preventing infection by specific viruses causing cervical cancer in young women (Villa et al., 2005). The current commercialized method of manufacturing VLP products relies on expression of the viral structural proteins in a eukaryotic cell system (e.g., in insect or yeast cell). On expression in the host cells, these proteins form VLPs within the cell interior which at the same time encapsulate host DNA fragments (Palkova et al., 2000). Studies have also indicated that the product from this manufacturing route consists of VLPs of varying particle size (Sasnauskas et al., 2002). The need to eliminate product heterogeneity and host impurities through complex purification has limited the capability of the current process to delivery VLP products quickly and at a minimal cost. In this work, we demonstrated the feasibility and effectiveness of processing VLPs in a cell-free reactor. This method relied first on the manufacture and purification, at high precision and homogeneity, of the protein subunits that make up the VLPs. Subsequent self-assembly processing of these subunits into VLPs was achieved through controlled changes in the physico-chemical environment containing the subunits. Since the VLPs were assembled from subunits of high quality, the resulting VLPs were more homogenous in terms of composition and architecture. Also, the complex and inefficient purification steps to remove similarly sized nanoparticles and intraparticle contaminants were avoided. Furthermore, an inexpensive and easily cultivated expression host such as Escherichia coli was used which would reduce the overall production cost significantly. State-of-the-art characterization techniques such as field-flow fractionation and multi-angle light scattering have been used in our study to quantitatively assess the quality of assembled VLPs, thus allowing us to link self-assembly process conditions to the final physical characteristics of the VLPs. Our results pave the way for a simple and economical way to produce VLPs with increased process efficiency and ease of optimization.

PALKOVA, Z., ADAMEC, T., LIEBL, D., STOKROVA, J. & FORSTOVA, J. (2000) Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures. Febs Letters, 478, 281-289.

SASNAUSKAS, K., BULAVAITE, A., HALE, A., JIN, L., KNOWLES, W. A., GEDVILAITE, A., DARGEVICIUTE, A., BARTKEVICIUTE, D., ZVIRBLIENE, A., STANIULIS, J., BROWN, D. W. G. & ULRICH, R. (2002) Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae. Intervirology, 45, 308-317.

VILLA, L. L., COSTA, R. L. R., PETTA, C. A., ANDRADE, R. P., AULT, K. A., GIULIANO, A. R., WHEELER, C. M., KOUTSKY, L. A., MALM, C., LEHTINEN, M., SKJELDESTAD, F. E., OLSSON, S. E., STEINWALL, M., BROWN, D. R., KURMAN, R., RONNETT, B. M., STOLER, M. H., FERENCZY, A., HARPER, D. M., TAMMS, G. M., YU, J., LUPINACCI, L., RAILKAR, R., TADDEO, F. J., JANSEN, K. U., ESSER, M. T., SINGS, H. L., SAAH, A. J. & LUPINACCI, L. (2005) Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncology, 6, 271-278.