156 Large-scale transient expression from mammalian cells is a new technology. Breakthroughs have been achieved for non-viral delivery methods: transfections can now be done at the 1–10 L scale with mammalian cells grown in suspension. Production of 1–20 mg/L of recombinant protein have been obtained in stirred bioreactors. Modified alphaviruses have provided a fast and efficient expression technology based on viral vectors. Addresses *Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland † Serono Pharmaceutical Research Institute, Geneva, Switzerland Current Opinion in Biotechnology 1999, 10:156–159 http://biomednet.com/elecref/0958166901000156 © Elsevier Science Ltd ISSN 0958-1669 Abbreviations HEK human embryonic kidney PEI polyethyleneimine SFV Semliki Forest virus Introduction Stable expression technologies, based on chromosome- integrated plasmid sequences, have delivered kilograms of complex proteins from mammalian cells. The devel- opment of these technologies requires considerable investment in time, human resources and bioreactor equipment. Due to high demand, the need for faster and cheaper approaches for the production of recombinant products is evident. The consideration of transient sys- tems for production of milligram to gram quantities of protein is rather new. With better technologies for reli- able growth of mammalian cells and with better nucleic acid transfer systems, the opportunity arose to explore transient expression in mammalian cells beyond the lab- oratory scale. In addition, many companies in the field of somatic gene therapy, using artificial or modified virus vectors, depend on transient DNA transfer to mam- malian cells as one of the key manufacturing steps for their products [1]. In 1997 and 1998 sessions at scientific meetings and even one entire meeting held in May 1997 had a focus on tran- sient gene expression in animal cells [2]. Here, we will review in a cursory way some of the most promising and successful approaches for scalable or large-scale transient gene expression in mammalian cells. The available meth- ods can be classified according to the vectors used: plasmid vectors or virus vectors. Key features of most transient expression systems are: 1. Simplicity, in particular in the construction of expres- sion vectors; 2. Extremely short time-frame for the generation of product (days); 3. Intrinsic genetic stability and consistency due to extremely short time-frame between generation of vector and product recovered; 4. Applicability to a wide range of host cell lines; 5. Suitability to multiple processing, allowing, study of many genes or mutants at the same time. Plasmid vectors General process A large number of DNA delivery methods for plasmids have been developed for mammalian cells. Only three have shown promise up to today for scalable operations: calcium phosphate DNA co-precipitation [3]; polyethyl- eneimine (PEI) DNA complexes [4]; and electroporation [5]. These three technologies have the advantage of being cost-effective. More importantly, they are suitable for both adherent and suspension cells. For cells grown in suspen- sion, these approaches have been used at scales up to ~10 L. Expression levels of 1–10 mg/L of product have been reported for secreted proteins, corresponding to spe- cific productivities of about 0.1–1 pg/cell/day. Common to these processes is the generation of rather large cell populations prior to DNA transfer. After trans- fection or electroporation, cells are maintained under production conditions for a limited time span, usually 5–10 days. The quantities of required plasmid range from 0.2 to 2 mg per 10 9 cells. Longer product accumulation periods can be used with vectors that are competent for episomal replication. Transfections or virus-mediated nucleic acid transfers with cells grown in suspension are promising for process scales beyond 10 L. A number of articles show that the basic principles of the technology are established or are being developed [6,7 •• ,8]. For mammalian cells that grow on substrates, the use of stacked multilayer systems have become very popular. Cells can be maintained in these production vessels for a limited period in static batch-type culture or for extended periods in a perfused mode. The disadvantage of stacked multilayer systems is cost; however, they have become a first choice approach in the field of somatic gene therapy for mass production of virus-based DNA vectors. Cell hosts Three cell types dominate the field: human embryonic kidney (HEK)-293, COS and baby hamster kidney (BHK) cells. Surprisingly, Chinese hamster ovary (CHO) cells, Large-scale transient expression in mammalian cells for recombinant protein production Florian Wurm* and Alain Bernard †