A Microscale Yeast Cell Disruption Technique for Integrated Process Development Strategies

Abstract

Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) viruslike particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (e8°C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.

Extraction of β-glucan from Saccharomyces cerevisiae: Comparison of different extraction methods and in vivo assessment of immunomodulatory effect in mice

“…Several extraction techniques are there to recover the potent BG from microbial and plant sources, but the choice of suitable extraction method plays a vital role that will affect the structure, quality, and functionality of the BG (Ahmad et al, 2012 from cytoplasm) and BG extraction (extraction from insoluble cell wall) (Javmen et al, 2012). Several studies reported about chemical (NaDH, HCl, acetic acid, citric acid) (Lee et al, 2001;Hunter et al, 2002;Pelizon et al, 2005), physical (sonication, high pressure) (Boonraeng et al, 2000;Shokri et al, 2008;Wenger et al, 2008) and enzymatic (lytic enzymes) lysis of yeast cells (Ferrer, 2006).…”

Design of the thermal insulation porous materials based on technogenic mineral fillers

“…Results of the studies by authors of [22,23] (on the ability of alkali-silicate systems with Al 2 O 3 to form insoluble products of Na 2 O·Al 2 O 3 ·2Si 2 O 3 ·nH 2 O in alkaline media) specify aluminum oxide of ash as a modifying component. It provides raw mixture with qualities required for the targeted product creation.…”

Formation of the steam phase in superheated liquids in the state of metastable equilibrium

“…We p F R W (13) As is known, the capillary force F σ regardless the shape of the deformed droplet always restores the spherical shape and the minimum Gibbs surface energy (steady state) is reached. Therefore, if the drop is deformed in the direction of its motion to a flattened ellipsoid, the capillary force counteracts the external force.…”

“…For example, in a water-oil type emulsion, interfacial tension is σ wo ≈16 mN/m, and tension at the water-vapor interface σ wv ≈73 mN/m [13]. At the same time, tension at the water-vapor interface with microscopic oil additives decreases to a value of σ wv ≈39...41 mN/m.…”