Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production.
Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5 alpha-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80 degrees C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.
Danquah
Michael K
MK
BEL (Bio Engineering Laboratory), Department of Chemical Engineering, Monash University, Wellington Road, Melbourne, VIC 3800, Australia. danquah@eng.monash.edu.au
Forde
Gareth M
GM
D002852:Chromatography, Ion Exchange / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D008689:Methacrylates / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D014462:Ultrafiltration / Q000379:methods
D004269:DNA, Bacterial; D008689:Methacrylates