Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

Abstract

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid-envelope virus-like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving approximately 70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of approximately 80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions ( approximately 400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low-shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two-fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan.

Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties

“…Usually, the nonionic detergent Triton X-100 is employed for solubilization of HBsAg form yeast homogenates, e.g. [9,13,45,46]. However, there have been early indications that the nonionic detergent Tween 20 might be less harsh to "intact" HBsAg compared to Triton X-100 [6].…”

A monolith purification process for virus-like particles from yeast homogenate

“…Cultures were grown in three stages; stages 1 and 2 were in shake flasks on a rotary shaker for 24 h and stage 3 was in a 75 L fermenter (Inceltech High containment fermenter, Maidenhead, UK) for 72 h. The full process and media components are Flow sheet of primary purification and pre-chromatography preparation steps adapted from [32]. The VLP is produced as intracellular product in recombinant Saccharomyces cerevisiae and the detergent Triton X-100 cleaves it off the endoplasmic reticulum.…”

“…2 and was adapted from Kee et al [32], Frozen cell paste was resuspended at 25% (w/v) in 0.1 M sodium phosphate, 0.5 M sodium chloride and 2 mM phenylmethylsulfonyl fluoride (dissolved in isopropanol). Cell disruption was carried out in a homogeniser at 1200 bar for three passes (Gaulin Micron Lab 40, APV Gaulin GmbH, Germany) or 400 bar for eight passes (Lab 60).…”

Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development

“…One of the antigens frequently used for VLP-based vaccine production is HBsAg. HBsAg has been expressed in P. pastoris, S. cerevisiae and Hansenula polymorpha [53][54][55][152][153][154][155]. Although high-level retention and accumulation of HBsAg in the ER have been documented, neither secretion of the protein nor intracellular VLP formation were observed, suggesting that VLP assembly took place during downstream processing of yeast biomass [62].…”