Large-scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA.
The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.
Kong
Simyee
S
Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
Rock
Cassandra F
CF
Booth
Andrew
A
Willoughby
Nicholas
N
O'Kennedy
Ronan D
RD
Relton
Julian
J
Ward
John M
JM
Hoare
Mike
M
Levy
M Susana
MS
D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation / Q000379:methods*; D004249:DNA Damage; D004278:DNA, Superhelical / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D005285:Fermentation; D005615:Freezing; D008970:Molecular Weight; D010957:Plasmids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D004278:DNA, Superhelical