Understanding the Chromatographic Processing of Recombinant Human Papillomavirus Vaccine Using Ceramic Hydroxypatite
Wang, Yiran, Chemical Engineering - School of Engineering and Applied Science, University of Virginia
Carta, Giorgio, Department of Chemical Engineering, University of Virginia
Virus like particles (VLPs) are novel biopharmaceutical products that have become widely used for vaccines, gene therapy and immunotherapy. The purification of VLPs poses several unique challenges including the handling of their large molecular size and the removal of nucleic acid contaminants. Ceramic hydroxyapatite (CHT), a mixed-mode chromatography adsorbent, has shown promise in purifying VLPs from nucleic acid due to its multimodal adsorption mechanism. This work has two principal objectives. The first is to investigate the interaction of RNA with VLPs using biolayer interferometry (BLI) measurements. Biotin tagged-RNA is immobilized on a streptavidin-coated biosensor and the VLP is immobilized through carboxyl-amine electrostatic interaction. The BLI observations show that VLP variants have different RNA association responses, which are also dependent on the buffer composition. The second objective focuses on characterizing CHT and evaluating its performance as a possible candidate for purifying VLPs and RNA removal. Confocal laser scanning microscopy (CLSM) with fluorescent labeling was used to visualize the adsorption behavior of both components. This work helps elucidates the mechanism of RNA association with VLPs and facilitates the design of new strategies for the next generation of VLP purification processes.
MS (Master of Science)
HPV, Virus like particles, Protein Chromatography, RNA–protein interaction, Biolayer Interferometry
All rights reserved (no additional license for public reuse)