Impact of the physical and topographical characteristics of adsorbent solid-phases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates.
A comparison is made of the performance of two types of adsorbent solid phases (commercially sourced Streamline composites and custom-assembled Zirblast pelliculates), derivatised with similar anion exchange chemistries and applied to the recovery of plasmid DNA from Escherichia coli extracts prepared by chemical lysis and coarse filtration. Streamline and Zirblast adsorbents were characterised by average particle diameters of 200 and 95 microm, densities of 1.16 and 3.85 g/m2, and small ion capacities of 170 and 8 micromol/ml settled adsorbent, respectively. Detailed analysis of products and impurities in a full operational cycle of adsorption, washing, pre-elution, elution and regeneration processes was enabled by the harnessing of a battery of analyses for nucleic acid and organic solute content of feedstocks and bed effluents exploiting ultra-violet spectrophotometry, agarose gel electrophoresis and specific reactions with the fluorescent probe PicoGreen. In comparative tests operated under near identical conditions, Streamline and Zirblast adsorbents exhibited plasmid recoveries of 76 and 90% of bound product characterised by purity ratios (relative PicoGreen and A254 estimates of mass) of 9 and 32, respectively. Conclusions are drawn regarding the specific impact of the physical and topographical characteristics of solid-phase geometry upon product throughput, achievable product purity, process time-scales and operational economics for the manufacture of plasmid DNA.
Thwaites
Eric
E
Biosystems Laboratory of the Research Centre for Formulation Engineering, School of Engineering, University of Birmingham, Edgbaston, UK.
Burton
Simon C
SC
Lyddiatt
Andrew
A
D000327:Adsorption; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids
D004269:DNA, Bacterial