Transient Gene Expression: A Novel Mammalian Cell-Based Technology for Recombinant Protein Production

Abstract

Methods for the transfection of DNA into mammalian cells for transient gene expression were initially developed about 30 years ago. The main purpose was and still remains the molecular analysis of the cell’s response to the exogenous protein. The use of transient gene expression for industrial purposes began in 1991 when Bennett and coworkers identified by alanine-scanning mutagenesis several mutants of human tissue-plasminogen activator (rtPA) that were eventually shown to be of higher medical potency than the wild-type protein. One of the molecules, now an approved thrombolytic “TNKase™”, had a dramatically extended half-life in patients, a high fibrin-specificity, and was insensitive to the inhibitor of plasminogen-activator PAI. The identification of improved mutants was based on more than 40,000 transient gene expression experiments (Wurm et al. unpublished) executed during the course of one year using calcium-phosphate transfections in human embryonic kidney (HEK) 293 cells (Gorman et al 1990).

Only very recently has the usefulness of transient gene expression for the production of milligram or even gram quantities of protein been recognized. A number of insights and technological advances were necessary to permit transient gene expression in mammalian cells to be executed in operational scales up to 100 Liters. Only two systems have so far been developed for this scale, calcium-phosphate based transient transfection of HEK-293 cells (Girard et al. 2002) and a Genentech-proprietary lipid based system using genetically modified Chinese hamster ovary (CHO) cells. This paper will summarize in a cursory way the most important developmental steps and the underlying arguments that allowed transient gene expression to become a second way for industrial protein production in mammalian cells. By comparison, virus based transient expression systems (Baculovirus, Alphavirus) have been restricted up to now to operational scales below 10 Liters and may not advance much further in terms of scale and high-throughput activity because of a number of inherent complexities, the most restrictive being a limit on the size/quantity of DNA that can be delivered. Other technological and biological problems also limit scale-up.