High-yield and scalable cell-free assembly of virus-like particles by dilution

Virus-like particles (VLPs) have been developed as safe and efficacious vaccines, and are increasingly used as carriers for foreign peptide epitopes in modular architectures. An emerging technology for low-cost and rapid-response VLP vaccine manufacture is based on controlled cell-free assembly of capsomeres, which are expressed in Escherichia coli, into VLPs presenting pharmaceutically relevant antigenic modules. A key bioprocessing challenge in this technology is VLP self-assembly, which has until now been studied using qualitative laboratory methods and without sufficient quantitation. In this work, the yield and size distribution of VLPs assembled by dialysis or by ten-fold dilution were compared using quantitative metrics. Membrane-based steps used for dialysis and particle concentration were identified as the key inefficiencies in each method, resulting in 13–18% protein loss. Key inefficiencies were circumvented through process intensification that led to development of a two-fold dilution assembly method. The new process eliminated a unit operation, improved the final concentration of assembly products by a factor of five and reduced buffer consumption nine-fold. Using this process, modular capsomeres presenting a group A Streptococcus (GAS) antigenic module were assembled into high-quality VLPs with a final yield of 54%, which was 18–22 percentage points higher than obtained using conventional methods described in the literature. This study demonstrates the feasibility of manufacturing VLP vaccines in cell-free reactors at high yield, with high structural integrity, using a scalable and simple process. Ongoing development based on these results will be conducive to process-intensified VLP bioprocessing for low-cost vaccine delivery at global scale.

► VLP yield and quality by dialysis and dilution assembly compared quantitatively.
► Process intensification leading to unit operation elimination.
► New process reduces buffer use 9× and increases product concentration 5×.
► VLP yield improved to 54%, 22 percentage points more than conventional methods.
► High-quality VLPs assembled using this scalable, high-yield cell-free method.