Plasmids are covalently closed, double stranded, DNA molecules which carry genetic information. These macromolecules have been used for gene therapy and DNA vaccination, and belong to a new class of medicinal agents that contain genetic materials. The manufacturing of plasmids under current Good Manufacturing Practices (cGMP) compliance as required by the Food and Drug Administration (FDA) and European Medicines Evaluation Agency (EMEA) is crucial to obtain a product which is consistent in purity, potency, identity, efficacy and safety. This paper gives an overview of the manufacturing of plasmids and provides the basic concepts for designing flowsheets for recovery and purification. The focus is mainly on the downstream processing, which is designed to release plasmid molecules (less than 1% w/w) from the Escherichia coli host cells and to remove impurities such as genomic DNA, RNA, proteins and endotoxins until the desired level of purity and other specifications are met. Many of the host impurities have similar properties to plasmid DNA, and will thus behave identically in most of the downstream processing steps. This constraints the selection and ordering of the units-operations used for separation throughout the process. The downstream processing unit operations can be grouped in three different stages: primary recovery, intermediate recovery and final purification. The objective of each of these stages as well as the unit operations more commonly used is described. The specific problems encountered that are associated with the structural nature of plasmids (high molecular weight, charge and flexibility) are highlighted. Four flowsheets that are representative of the current panorama in downstream processing of plasmid DNA are described and their weaknesses highlighted. An alternative process solution is suggested that, although unproven, addresses many of the weaknesses of the existing processes. (C) 2003 Elsevier B.V. All rights reserved.