A microscale yeast cell disruption technique for integrated process development strategies.
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) virus-like 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 (< =8 degrees 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.
Wenger
Marc D
MD
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. marc_wenger@merck.com
DePhillips
Peter
P
Bracewell
Daniel G
DG
D002458:Cell Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D008832:Microchemistry / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*; D013010:Sonication; D018511:Systems Integration
D029701:Saccharomyces cerevisiae Proteins