In recent years, the development of novel recombinant virus-like particles (VLPs) has been generating new perspectives for the prevention of untreated and arising infectious diseases. However, cost-reduction and acceleration of manufacturing processes for VLP-based vaccines or vectors are key challenges for the global health system. In particular, the design of rapid and cost-efficient purification processes is a critical bottleneck. In this review, we describe and evaluate new concepts, development strategies and unit operations for the downstream processing of VLPs. A special focus is placed on purity requirements and current trends, as well as chances and limitations of novel technologies. The discussed methods and case studies demonstrate the advances and remaining challenges in both rational process development and purification tools for large biomolecules. The potential of a new era of VLP-based products is highlighted by the progress of various VLPs in clinical phases.

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A c c e p t e d M a n u s c r i p t -Establishing a high-throughput procedure for miniaturized, parallel and automated cultivations, cell lysis and analytics of recombinant virus proteins -Optimizing the production of a promising virus-like particle-based nanocarrier for future chimeric vaccines in Escherichia coli -Yielding up to 1.63 mg of murine polyomavirus protein VP1 per mL cultivation medium with a total soluble protein portion of 43% by codon optimization and design of experiments -Developing a simple membrane-based two-step downstream process for the murine polyomavirus protein VP1 -Assembling murine polyomavirus protein VP1 into homogeneous VLPs with a size of 40-50 nm and a protein purity of 92% AbstractThe production of safe vaccines against untreatable or new diseases has pushed the research in the field of virus-like particles (VLPs). Currently, a large number of commercial VLP-based human vaccines and vaccine candidates are available or under development. A promising VLP production route is the controlled in vitro assembly of virus proteins into capsids.In the study reported here, a high-throughput screening (HTS) procedure was implemented for the upstream process development of a VLP platform in bacterial cell systems. Miniaturized cultivations were carried out in 48-well format in the BioLector system (m2p-Labs, Germany) using an Escherichia coli strain with a tac promoter producing the murine polyomavirus capsid protein (VP1). The screening procedure incorporated micro-scale cultivations, HTS cell disruption by sonication and HTS-compatible analytics by capillary gel electrophoresis. Cultivation temperatures, shaking speeds, induction and medium conditions were varied to optimize the product expression in E. coli. The most efficient system was selected based on an evaluation of soluble and insoluble product concentrations as well as on the percentage of product in the total soluble protein fraction. The optimized system was scaled up to cultivation 2.5 L shaker flask scale and purified using an anion exchange chromatography membrane adsorber, followed by a size exclusion chromatography polishing procedure. For proof of concept, purified VP1 capsomeres were assembled under defined buffer conditions into empty capsids and characterized using transmission electron microscopy (TEM).The presented HTS procedure allowed for a fast development of an efficient production process of VLPs in E. coli.Under optimized cultivation conditions, the VP1 product totalled up to 43% of the total soluble protein fraction, yielding 1.63 mg VP1 per mL of applied cultivation medium. The developed production process strongly promotes the murine polyoma-VLP platform, moving towards an industrially feasible technology for new chimeric vaccines.