Optimization of Isopropanol and Ammonium Sulfate Precipitation Steps in the Purification of Plasmid DNA S. S. Freitas, J. A. L. Santos, and D. M. F. Prazeres* Centro de Engenharia Biolo ´gica e Quı ´mica, Instituto Superior Te ´cnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 μg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 μg pDNA/ mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 °C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (≈100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process. Introduction Gene therapy has been heralded as a new and promising approach for the treatment of genetic disorders and acquired diseases. Typically, it involves the use of a vector such as plasmid DNA for the delivery of a therapeutic gene to specific target cells. Although gene therapy was originally proposed for the treatment of single-gene disorders, the majority of current clinical trials involve the treatment of cancer and vascular disease (1). In addition, the technique is also being developed to provide DNA vaccines for infectious diseases (2, 3). Despite the potential demonstrated, nonviral gene therapy and DNA vaccinations are still under development, and a therapeutic product or protocol has yet to reach the market. In general, pure plasmid vectors required for a DNA vaccine and gene therapy studies are purified using commercial kits developed for laboratory bench-scale applications. However, the increasing number of applications that are moving from the laboratory to the clinic is creating a need for large amounts of highly purified plasmid DNA. This demand is expected to increase as the first products reach the market. Thus, there is a clear need for a parallel development of efficient, scalable, and reproducible plasmid DNA manufacturing processes capable of delivering high amounts of product (4-6). Precipitation is a relatively simple and inexpensive technique often used as an intermediate recovery step in the downstream processing of plasmid DNA. The starting point is usually a complex and viscous lysate obtained after cell lysis. The plasmid in this stream is usually diluted, and although most genomic DNA (gDNA) has been removed, large amounts of RNA and proteins, which account for more than 90% of the total solute mass, still remain in the clarified lysates (7, 8). Major goals for precipitation are, thus, to remove impurities and/or concentrate the plasmid product. Concentration is commonly performed by precipitating the plasmid with agents such as 2-propanol (9), poly(ethylene glycol) (10), spermidine (11), or cetyl trimethyl- ammonium bromide (CTAB) (12). The removal of low molec- ular weight nucleic acids and the possibility of performing a buffer exchange are additional advantages of this operation. And, in the case of CTAB, an adequate modulation of the precipitation conditions additionally offers a selectivity degree that is high enough to separate open circular from supercoiled plasmid isoforms (12). After the concentration of plasmid by precipita- tion, a large fraction of impurities (proteins, endotoxins, and higher molecular weight RNA) can be removed with an additional precipitation step using antichaotropic salts such as * To whom correspondence should be addressed. Phone: + 351 218419133. Fax: + 351 218419062. E-mail: prazeres@alfa.ist.utl.pt. 1179 Biotechnol. Prog. 2006, 22, 1179-1186 10.1021/bp060052d CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/06/2006