title: High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli.
abstract: The 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 2.5L 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.
authors.affiliation: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: christopher.ladd@kit.edu.
authors.forename: Christopher
authors.initials: C
authors.lastname: Ladd Effio
authors.affiliation: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: pascal.baumann@kit.edu.
authors.forename: Pascal
authors.initials: P
authors.lastname: Baumann
authors.affiliation: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Weigel
authors.affiliation: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Vormittag
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia.
authors.forename: Anton
authors.initials: A
authors.lastname: Middelberg
authors.affiliation: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D046529:Microscopy, Electron, Transmission; D013010:Sonication; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis* / Q000302:isolation & purification*
chemical_list: D036022:Capsid Proteins; C062481:VP1 protein, polyomavirus; D058425:Vaccines, Virus-Like Particle
keywords: BioLector; Escherichia coli; High-throughput screening; Micro-scale cultivation; Virus-like particles