Virus-like particles (VLPs) are supramolecular protein assemblies with applications in medicine and biotechnology, yet their potential is limited by poorly scalable manufacturing and inconsistent product architectures. Cell-free protein synthesis (CFPS) offers an alternative approach for VLP production. Cell-free extracts from a variety of host organisms have been explored to exploit advantageous properties intrinsic to those organisms. The yeast Pichia pastoris is a particularly attractive eukaryotic host for CFPS due to its genetic tractability, versatility, ability to perform post-translational modifications, and use as a VLP manufacturing host.
This work focuses on development of a Pichia pastoris CFPS system as a prototyping tool for VLPs. This project is divided in to four sections: 1) Further development of the P. pastoris CFPS system, 2) Enhancing the productivity of the system using a Design of Experiments (DOE) approach, 3) Demonstrating the capability of the platform by producing the model Hepatitis B core (HBc) antigen VLP, 4) Demonstrating the versatility of the system by producing Human papillomavirus (HPV) VLPs from multiple genotypes.
Reformulation of the complex reaction mixture was achieved using a DOE approach, leading to a 3.5-fold increase in synthesis of the biopharmaceutical human serum albumin and a 4.8-fold increase in luciferase expression compared to previously established conditions. The potential of the platform for VLP production and prototyping was demonstrated by developing a rapid workflow for the crude purification and imaging of HBc VLPs. Synthesized particles were found to have comparable characteristics to those previously produced in vivo and in vitro. A workflow was then developed to characterise morphological changes in HPV VLPs as a result of genotype, assembly environment conditions, and codon usage. The versatility of the platform suggests the P. pastoris system could be used to prototype novel vaccine candidates and pre-screen genetic constructs prior to in vivo expression.