High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli

Abstract

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.

Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures

“…Several studies have shown the results of induction profiles in 48-well Flowerplates but either at single temperatures [ 24 , 30 – 32 ] or with few data points [ 33 , 34 ]. The aim of this study was to comprehensively investigate the effect of temperature on optimal induction conditions.…”

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

“…An eight‐well sonifyer for the release of virus‐like particles from E. coli was used by Ladd et al. . Another physical HT cell breakage system based on electric fields in parallel microfluidic channels was developed by Poudineh et al.…”

“…A 24-well HT sonication device was used by Hohnadel et al [76] for 15 distinct cell types including gram-negative and -positive bacteria as well as fungi and yeasts. An eight-well sonifyer for the release of virus-like particles from E. coli was used by Ladd et al [77]. Another physical HT cell breakage system based on electric fields in parallel microfluidic channels was developed by Poudineh et al [78].…”

Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden

“…A popular noninvasive online‐monitoring device for microtiter plate cultivations is the BioLector device. Biomass and fluorescent products can simply be measured via scattered light (ScL) and fluorescence intensities, respectively . A decisive advantage is the noninvasive procedure, which allows measurement of samples without stopping the shaking process of the microtiter plate .…”

“…Biomass and fluorescent products can simply be measured via scattered light (ScL) and fluorescence intensities, respectively. [22][23][24][25][26][27][28][29] A decisive advantage is the noninvasive procedure, which allows measurement of samples without stopping the shaking process of the microtiter plate. 29 By using special microtiter plates equipped with optodes, also oxygen or pH can be detected.…”