Large target molecules (> Mr 700 000) pass outside the bead, while smaller molecules can bind to the inner core (Fig 2). The bead has octylamine ligands coupled to the core that is surrounded by an inert outer layer. Capto Core 700 represents a new generation of chromatography media and is designed and optimized for purification of viruses and other large biomolecules. Protein purity was calculated as the L1 protein-to-total protein ratio and expressed as a percentage. The tentative purity target was set to > 95% as determined by densitometry analysis of electrophoresis gels. Here, we describe a simple procedure to separate the VLPs from host cell-derived impurities to high purity using two orthogonal chromatographic steps: layered-bead size exclusion and anion exchange chromatography. To reach adequate purity, previously established purification processes combines anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. Five L1 protein units form pentamers, which in turn self-assemble into VLPs (Fig 1). Here, the structural L1 protein is expressed in insect Sf9 cells using a baculovirus vector. Nevertheless, the vaccine triggers an immunogenic response that protects against HPV infection. The VLP vaccine lacks the viral nucleic acid and, hence, cannot induce infection. L1 proteins spontaneously self-assemble to hollow VLPs that resemble authentic HPV virons. As papillomaviruses are not easily grown in cell cultures, the vaccine constructs are VLP-based, most commonly with recombinant L1 protein as the structural component. In this work, HPV 58, a subtype commonly occurring in Asia (2), was used as vaccine immunogene. However, vaccines protecting against more genotypes would prevent more cancer forms and be less susceptible to regional variations of the virus. Current vaccines protect against up to four virus genotypes. To prevent cervical cancer and to reduce the number of treatments for cervical cancer precursors, the World Health Organization (WHO) recommends vaccination of young women against HPV (1). Several genotypes are also classified as low risk and associated with benign lesions and genital warts. More than 111 genotypes of HPV have been described, among which about 30 have been associated with anogenital cancers. HPV is a common sexually transmitted infection in adults. In the second step, Capto Q ImpRes medium allows polishing of the L1 protein with high purity and resolution. In the first step, VLPs are purified in flow-through mode using Capto Core 700, a layered-bead size exclusion medium. A modern, scalable approach, based on two chromatographic steps including novel chromatography media (resins), was used for the purification of L1 protein. Modified L1 protein was expressed in insect Sf9 cells using baculovirus vector, and allowed to spontaneously assemble into virus like particles (VLPs). This application note describes the results of a project executed by the GE Healthcare BioProcess™ Services team to display proof of principle of a purification procedure for human papillomavirus (HPV) L1 protein, the antigenic component of HPV vaccines.
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