Development of the initial phase of a DNA plasmid purification process ; DNA-plasmidin puhdistusprosessin alkuvaiheen kehittäminen

The objective of this work was to compare chromatographic procedures in plasmid purification as well as to develop the lysis stage of the purification process. Plasmid containing bacterial pellet was lysed by an alkali procedure. Precipitate that was generated during the lysis stage and which contained mainly impurities was removed by centrifugation. Chromatographic procedures were utilized for purification of plasmid DNA. Six different chromatography columns (ii Prep 16/10 ANX FF (high sub), Poros 50 P1, Poros 50 D, Sartobind D 75, CHT Ceramic Hydroxyapatite Type II, Capto DEAE) were compared with a well established plasmid purification procedure (Qiagen EndoFree Plasmid Mega Kit). The results were evaluated on the basis of product purity, process yield and time consumption. Product purity was investigated for endotoxins, RNA, genomic DNA, proteins and plasmid homogeneity. The centrifugation in lysis stage was replaced with filtration and obtained results were compared with the original process. Of the evaluated chromatographic procedures Sartobind D 75 -membrane was found to be best. Product purity was superior in comparison with the other procedures. Process yield was 40 % which was comparable to the established plasmid purification procedure. In addition Sartobind D 75 -membranes were fast and convenient to use. Results from Capto DEAE -column was very promising. Process yield was 63 % which was 20 % higher than the established plasmid purification process. The product purity should be further investigated. Process yield was below 30 % for HiPrepl6/10 ANX FF (high sub) and Poros 50 P1 columns. The product was also contaminated with significant amounts of RNA. The product purity of CHT Ceramic Hydroxyapatite Type II -column was insufficient according to all criteria. The procedure was also more labour-consuming than the others. Poros 50 D -column did not purify the plasmid in the process conditions. The filters which were used to replace centrifugation were found to be able to remove precipitate from the ...