Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications

Human clinical trial of gene therapy with nonviral vectors demands large am ounts of pharmaceutical:grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the developme nt of processing methodologies for the large-scale production and purificat ion of plasmids. This work describes several studies that were undertaken d uring the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine techniqu e for monitoring plasmid purity in process streams. The use of RNase or hig h temperatures during alkaline lysis was proved unnecessary. Instead, RNA c ould be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusio n chromatography. Also, clarification of streams by precipitation was indep endent of the chaotropic salt used. Furthermore, by proceeding directly fro m cell lysis to chromatography it was possible to obtain plasmid with purit y/quality identical to that of the one obtained when clarification and conc entration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-der ived enzymes, alcohols, or toxic solvents were used, rendering validation p otentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without a ffecting plasmid purity.