Development of Process Flow Sheets for the Purification of Supercoiled Plasmids for Gene Therapy Applications Guilherme N. M. Ferreira, Joaquim M. S. Cabral, and Duarte M. F. Prazeres* Centro de Engenharia Biolo ´gica e Quı ´mica, Instituto Superior Te ´cnico, Avenue Rovisco Pais 1096 Lisboa Codex, Portugal Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodolo- gies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration 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-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity. Introduction One of the technological challenges associated with gene therapy is the development of large-scale processes for the production and purification of plasmid vectors capable of providing a safe, efficient, and cost-effective product (1), complying with the requirements of regula- tory agencies (2). Although plasmids are produced in Escherichia coli cells through a fermentation as most recombinant proteins, reports of processing methods for their large-scale production are scarce (3). As in the purification of proteins, plasmids must be isolated from host cells, the extract must then be clarified and concen- trated, after which the plasmids must be purified, and the final preparation should be polished. The challenge in plasmid purification is to remove impurities with similar properties, such as lipopolysaccharides, RNA, and chromosomal DNA, that behave identically in most of the above-mentioned steps (4). There is also a current understanding, although not clearly demonstrated and accepted, that plasmid vectors should be mostly in the supercoiled form, which is thought to be more effective in transferring gene expression (5). Protocols for plasmid isolation in molecular biology are time-consuming, expensive, not amenable to scale-up, and generally not safe for therapeutic purposes due to the use of toxic solvents, mutagenic reagents, and animal- derived enzymes such as bovine pancreatic RNase A. At process scale, the usual approach has been to use the standard alkaline lysis method (6, 7) followed by unit operations designed to meet product specifications (6). While most genomic DNA is denatured and precipitated during lysis (8), large amounts of RNA and proteins remain to be removed in subsequent steps. Though most of the published purification schemes suggest the use of RNase (9) or high temperatures (10) to reduce the content of high molecular weight RNA, the impact of using RNase in terms of process economics, approval, and validation has not been pondered, and the effect of high temperature together with harsh alkaline conditions on plasmid stability has not been fully addressed. Clarification and concentration after lysis by such methods as ammonium acetate and poly(ethylene glycol) precipitation are commonly used to further remove host proteins and small nucleic acids and to reduce the volume of process streams prior to chromatographic purification (6). Chromatography is relatively easy to optimize and scale-up, and several plasmid properties such as charge (11) and size (12) can be explored in the design of these separations. This work reports the development of two process flow sheets for the purification of supercoiled plasmid DNA based on alkaline lysis and chromatogra- phy (Figure 1). A 4.8 kb plasmid, pMa5-L, encoding a foreign gene (13) was used as a model system. Within the process, several options were considered mainly at the lysis and clarification stages. The effect of tempera- ture and RNase during lysis, as well as the effect of several chaotropic agents in the clarification stage, was * Corresponding author. Phone: +351-1-841 91 33. Fax: +351- 1-841 90 62. 725 Biotechnol. Prog. 1999, 15, 725-731 10.1021/bp990065+ CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/09/1999