Virus-like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, i.e. capsomeres, composed of recombinantly expressed viral structural proteins. During recombinant production, e.g. in Escherichia coli, VLPs usually self-assemble in host cells. Downstream processing of these in vivo-assembled VLPs typically includes VLP dis- and reassembly to remove encapsulated impurities and to improve structural homogeneity, stability, and immunogenicity. Disassembly is achieved in a high-pH solution and by addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material-consuming disassembly experiments. To this end, we developed a low-volume and high-resolution disassembly screening that provides time-resolved insight into the VLP disassembly progress. In this study, two variants of C-terminally truncated hepatitis B core antigen VLPs were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. In the second part of this work and based on the high-throughput screening results, a diafiltration-based disassembly process step was developed. Compared to mixing-based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration-based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high-molecular-weight species. The predominantly size-based separations in this approach promise a simple transfer to other chimeric VLP candidates or VLPs.

Process development for cross-flow diafiltration-based VLP disassembly: A novel high-throughput screening approach

ACS Fall 2021

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